Secondary literature sources for NDK

The following references were automatically generated.

Schneider B et al.
Activation of anti-reverse transcriptase nucleotide analogs by nucleoside diphosphate kinase: improvement by alpha-boranophosphate substitution.
Nucleosides Nucleotides Nucleic Acids. 2001; 20: 297-306
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Nucleoside activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively for a new class of nucleoside analogs with a borano (BH3-) or a thio (SH) group on the alpha-phosphate. Both the alpha-Rp-borano derivatives of AZT and d4T improved phosphorylation by NDP kinase, inhibition of reverse transcription as well as stability of alpha-borano nonophosphate derivatives in terminated viral DNA chain.

Zhou Y, Artman M
Nucleoside diphosphate kinase: a new player in heart failure?
Cardiovasc Res. 2001; 49: 7-10

Schneider B, Babolat M, Xu YW, Janin J, Veron M, Deville-Bonne D
Mechanism of phosphoryl transfer by nucleoside diphosphate kinase pH dependence and role of the active site Lys16 and Tyr56 residues.
Eur J Biochem. 2001; 268: 1964-71
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Nucleoside diphosphate (NDP) kinase phosphorylates nucleoside diphosphates with little specificity for the base and the sugar. Although nucleotide analogues used in antiviral therapies are also metabolized to their triphosphate form by NDP kinase, their lack of the 3'-hydroxyl of the ribose, which allows them to be DNA chain terminators, severely impairs the catalytic efficiency of NDP kinase. We have analyzed the kinetics parameters of several mutant NDP kinases modified on residues (Lys16, Tyr56, Asn119) interacting with the gamma-phosphate and/or the 3'-OH of the Mg2+-ATP substrate. We compared the relative contributions of the active-site residues and the substrate 3'-OH for point mutations on Lys16, Tyr56 and Asn119. Analysis of additional data from pH profiles identify the ionization state of these residues in the enzyme active form. X-ray structure of K16A mutant NDP kinase shows no detectable rearrangement of the residues of the active site.

Cervoni L et al.
Binding of nucleotides to nucleoside diphosphate kinase: a calorimetric study.
Biochemistry. 2001; 40: 4583-9
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The source of affinity for substrates of human nucleoside diphosphate (NDP) kinases is particularly important in that its knowledge could be used to design more effective antiviral nucleoside drugs (e.g., AZT). We carried out a microcalorimetric study of the binding of enzymes from two organisms to various nucleotides. Isothermal titration calorimetry has been used to characterize the binding in terms of Delta G degrees, Delta H degrees and Delta S degrees. Thermodynamic parameters of the interaction of ADP with the hexameric NDP kinase from Dictyostelium discoideum and with the tetrameric enzyme from Myxococcus xanthus, at 20 degrees C, were similar and, in both cases, binding was enthalpy-driven. The interactions of ADP, 2'deoxyADP, GDP, and IDP with the eukaryotic enzyme differed in enthalpic and entropic terms, whereas the Delta G degrees values obtained were similar due to enthalpy--entropy compensation. The binding of the enzyme to nonphysiological nucleotides, such as AMP--PNP, 3'deoxyADP, and 3'-deoxy-3'-amino-ADP, appears to differ in several respects. Crystallography of the protein bound to 3'-deoxy-3'-amino-ADP showed that the drug was in a distorted position, and was unable to interact correctly with active site side chains. The interaction of pyrimidine nucleoside diphosphates with the hexameric enzyme is characterized by a lower affinity than that with purine nucleotides. Titration showed the stoichiometry of the interaction to be abnormal, with 9--12 binding sites/hexamer. The presence of supplementary binding sites might have physiological implications.

Krishnan KS et al.
Nucleoside diphosphate kinase, a source of GTP, is required for dynamin-dependent synaptic vesicle recycling.
Neuron. 2001; 30: 197-210
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Nucleoside diphosphate kinase (NDK), an enzyme encoded by the Drosophila abnormal wing discs (awd) or human nm23 tumor suppressor genes, generates nucleoside triphosphates from respective diphosphates. We demonstrate that NDK regulates synaptic vesicle internalization at the stage where function of the dynamin GTPase is required. awd mutations lower the temperature at which behavioral paralysis, synaptic failure, and blocked membrane internalization occur at dynamin-deficient, shi(ts), mutant nerve terminals. Hypomorphic awd alleles display shi(ts)-like defects. NDK is present at synapses and its enzymatic activity is essential for normal presynaptic function. We suggest a model in which dynamin activity in nerve terminals is highly dependent on NDK-mediated supply of GTP. This connection between NDK and membrane internalization further strengthens an emerging hypothesis that endocytosis, probably of activated growth factor receptors, is an important tumor suppressor activity in vivo.

Kreimeyer A et al.
NDP kinase reactivity towards 3TC nucleotides.
Antiviral Res. 2001; 50: 147-56
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Nucleoside diphosphate (NDP) kinase is usually considered as the enzyme responsible for the last step of the cellular phosphorylation pathway leading to the synthesis of biologically active triphospho-derivatives of nucleoside analogs used in antiviral therapies and in particular in the treatment of AIDS. NDP kinase lacks specificity for the nucleobase and can use as substrate both ribo- or 2'-deoxyribonucleotides. However, only nucleoside analogs with a sugar moiety in the D-configuration (e.g. 3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T)) have so far been analyzed as substrates of NDP kinase. In contrast, beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), also called lamivudine, is a nucleoside analog that is now widely used in AIDS therapy and has a sugar moiety in the L-configuration. Using protein fluorescence to monitor the phosphotransfer between the enzyme and the nucleotide derivative at the presteady state, we have studied the reactivity of 3TC triphosphate and of other L-dideoxynucleotides with NDP kinase. We found that L-dideoxynucleoside triphosphates have a poor affinity for NDP kinase and that the catalytic efficiency of the phosphorylation of L-dideoxyderivatives is very low as compared with their D-enantiomers. We discuss these results using a computer model of 3TC diphosphate bound to the NDP kinase active site. NDP kinase may not seem to be the major enzyme phosphorylating 3TC-DP, in contrast to current opinion.

Gounaris K, Thomas S, Najarro P, Selkirk ME
Secreted variant of nucleoside diphosphate kinase from the intracellular parasitic nematode Trichinella spiralis.
Infect Immun. 2001; 69: 3658-62
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The molecular components involved in the survival of the parasitic nematode Trichinella spiralis in an intracellular environment are poorly characterized. Here we demonstrate that infective larvae secrete a nucleoside diphosphate kinase when maintained in vitro. The secreted enzyme forms a phosphohistidine intermediate and shows broad specificity in that it readily accepts gamma-phosphate from both ATP and GTP and donates it to all nucleoside and deoxynucleoside diphosphate acceptors tested. The enzyme was partially purified from culture medium by ATP affinity chromatography and identified as a 17-kDa protein by autophosphorylation and reactivity with an antibody to a plant-derived homologue. Secreted nucleoside diphosphate kinases have previously been identified only in prokaryotic organisms, all of them bacterial pathogens. The identification of a secreted variant of this enzyme from a multicellular eukaryote is very unusual and is suggestive of a role in modulating host cell function.

Galvis ML, Marttila S, Hakansson G, Forsberg J, Knorpp C
Heat stress response in pea involves interaction of mitochondrial nucleoside diphosphate kinase with a novel 86-kilodalton protein.
Plant Physiol. 2001; 126: 69-77
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In this work we have further characterized the first mitochondrial nucleoside diphosphate kinase (mtNDPK) isolated from plants. The mitochondrial isoform was found to be especially abundant in reproductive and young tissues. Expression of the pea (Pisum sativum L. cv Oregon sugarpod) mtNDPK was not affected by different stress conditions. However, the pea mtNDPK was found to interact with a novel 86-kD protein, which is de novo synthesized in pea leaves upon exposure to heat. Thus, we have evidence for the involvement of mtNDPK in mitochondrial heat response in pea in vivo. Studies on oligomerization revealed that mtNDPK was found in complexes of various sizes, corresponding to the sizes of e.g. hexamers, tetramers, and dimers, indicating flexibility in oligomerization. This flexibility, also found for other NDPK isoforms, has been correlated with the ability of this enzyme to interact with other proteins. We believe that the mtNDPK is involved in heat stress response in pea, possibly as a modulator of the 86-kD protein.

Ishibashi M et al.
NaCl-activated nucleoside diphosphate kinase from extremely halophilic archaeon, Halobacterium salinarum, maintains native conformation without salt.
FEBS Lett. 2001; 493: 134-8
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Enzymes from extremely halophilic archaea are readily denatured in the absence of a high salt concentration. However, we have observed here that a nucleoside diphosphate kinase prepared from Halobacterium salinarum was active and stable in the absence of salt, though it has the amino acid composition characteristic of halophilic enzymes. Recombinant nucleoside diphosphate kinase expressed in Escherichia coli requires salt for activation in vitro, but once it acquires the proper folding, it no longer requires the presence of salts for its activity and stability.

Erent M et al.
Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene.
Eur J Biochem. 2001; 268: 1972-81
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The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase CDelta. NDP kinase CDelta had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase CDelta, based on the crystal structure of NDP kinase B, indicated that NDP kinase CDelta had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase CDelta readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo.

Pan L, Kawai M, Yano A, Uchimiya H
Nucleoside diphosphate kinase required for coleoptile elongation in rice.
Plant Physiol. 2000; 122: 447-52
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Although several nucleoside diphosphate (NDP) kinase genes have been cloned in plants, little is known about the functional significance of this enzyme during plant growth and development. We introduced a chimeric gene encoding an antisense RNA of NDP kinase under the control of the Arabidopsis heat shock protein HSP81-1 promoter into rice (Oryza sativa L.) plants using the Agrobacterium tumefaciens transformation system. The expression of antisense RNA down-regulated the accumulation of mRNA, resulting in reduced enzyme activity even under the standard growth temperature (25 degrees C) in transgenic plants. Following heat shock treatment (37 degrees C), NDP kinase activities in some transgenic rice plants were more reduced than those grown under 25 degrees C. The comparison of the coleoptile growth under submersion showed that cell elongation process was inhibited in antisense NDP kinase transgenic plants, suggesting that an altered guanine nucleotide level may be responsible for the processes.

Hamby CV et al.
Expression of a catalytically inactive H118Y mutant of nm23-H2 suppresses the metastatic potential of line IV Cl 1 human melanoma cells.
Int J Cancer. 2000; 88: 547-53
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Nm23-H1 and nm23-H2 are putative metastasis suppressor genes that encode nucleoside diphosphate kinase (NDPK) A and B. NDPKs form oligomers distributed between soluble and particulate fractions of cells and therefore may exert their effects as either soluble or bound proteins. To determine whether metastasis-related functions of NDPKs are mediated by their catalytic activity in membrane bound or soluble complexes, we have stably transfected highly metastatic human melanoma Line IV Cl 1 cells with wild-type and catalytically inactive (H118Y) nm23-H1 and nm23-H2 genes and assayed their metastatic potential in nude mice. Transfection with wild-type nm23-H1 and nm23-H2 genes and catalytically inactive nm23-H1 did not significantly (all p > 0.10) alter the metastatic potential of Line IV Cl 1 cells while transfection with catalytically inactive nm23-H2 significantly (p < 0.01) reduced their metastatic potential. The lack of effect of transfection with wild-type and catalytically inactive nm23-H1 suggests that neither soluble nor membrane bound NDPK A affect the metastatic potential of Line IV Cl 1 cells. The metastasis suppressive effect of catalytically inactive NDPK B overexpression suggests that competition with bound complexes containing catalytically active NDPK B inhibits metastasis of Line IV Cl 1 cells. These results imply that bound NDPK B promotes metastasis and suggest that inhibition of its function or of its binding to critical sites may be a useful approach to limit the development of metastases in human melanoma.

Yano M
[Intrinsic nucleoside diphosphate kinase-type activity is a novel function of the molecular chaperone and protease]
Seikagaku. 2000; 72: 41-5

Abdulaev NG, Kakuev DL, Ridge KD
Bovine retinal nucleoside diphosphate kinase: biochemistry and molecular cloning.
Methods Enzymol. 2000; 316: 87-100

Postel EH, Abramczyk BM, Levit MN, Kyin S
Catalysis of DNA cleavage and nucleoside triphosphate synthesis by NM23-H2/NDP kinase share an active site that implies a DNA repair function.
Proc Natl Acad Sci U S A. 2000; 97: 14194-9
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NM23/NDP kinases play an important role in development and cancer but their biological function is unknown, despite an intriguing collection of biochemical properties including nucleoside-diphosphate kinase (NDP kinase), DNA binding and transcription, a mutator function, and cleavage of unusually structured DNA by means of a covalent enzyme-DNA complex. To assess the role of the nuclease in human NM23-H2, we sought to identify the amino acid responsible for covalent catalysis. By sequencing a DNA-linked peptide and by site-directed mutagenesis, we identified lysine-12, a phylogenetically conserved residue, as the amino acid forming the covalent complex with DNA. In particular, the varepsilon-amino group acts as the critical nucleophile, because substitution with glutamine but not arginine completely abrogated covalent adduct formation and DNA cleavage, whereas the DNA-binding properties remained intact. These findings and chemical modification data suggest that phosphodiester-bond cleavage occurs by a DNA glycosylase/lyase-like mechanism known as the signature of base excision DNA repair nucleases. Involvement of NM23/NDP kinase in a DNA repair pathway would be consistent with its role in normal and tumor cell development. Additionally, lysine-12, which is known in the x-ray crystallographic structure to lie in the catalytic pocket involved in the NDP kinase phosphorylation reaction, was found essential also for the NDP kinase activity of NM23-H2, suggesting that the two catalytic activities of NM23-H2 are fundamentally connected.

Milon L et al.
The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase.
J Biol Chem. 2000; 275: 14264-72
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We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.

Schneider B et al.
The mechanism of phosphorylation of anti-HIV D4T by nucleoside diphosphate kinase.
Mol Pharmacol. 2000; 57: 948-53
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The last step in the intracellular activation of antiviral nucleoside analogs is the addition of the third phosphate by nucleoside diphosphate (NDP) kinase resulting in the synthesis of the viral reverse transcriptase substrates. We have previously shown that dideoxynucleotide analogs and 3'-deoxy-3'-azidothymidine (AZT) as di- or triphosphate are poor substrates for NDP kinase. By use of protein fluorescence, we monitor the phosphotransfer between the enzyme and the nucleotide analog. Here, we have studied the reactivity of D4T (2',3'-dideoxy-2',3'-didehydrothymidine; stavudine) as di- (DP) or triphosphate (TP) at the pre-steady state. The catalytic efficiency of D4T-DP or -TP is increased by a factor of 10 compared with AZT-DP or -TP, respectively. We use an inactive mutant of NDP kinase to monitor the binding of a TP derivative, and show that the affinity for D4T-TP is in the same range as for the natural substrate deoxythymidine triphosphate, but is 30 times higher than for AZT-TP. Our results indicate that D4T should be efficiently phosphorylated after intracellular maturation of a prodrug into D4T-monophosphate.

Venturelli D, Cesi V, Ransac S, Engelhard A, Perrotti D, Calabretta B
The nucleoside diphosphate kinase activity of DRnm23 is not required for inhibition of differentiation and induction of apoptosis in 32Dcl3 myeloid precursor cells.
Exp Cell Res. 2000; 257: 265-71
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DRnm23 belongs to a multigene family which includes nm23-H1, the first bona fide metastasis suppressor gene, nm23-H2, nm23-H4, and nm23-H5. Like nm23-H1, nm23-H2, and nm23-H4, DRnm23 possesses nucleoside diphosphate kinase (NDPK) activity. Upon overexpression in myeloid precursor 32Dcl3 cells, DRnm23 inhibits granulocytic differentiation and promotes apoptosis. Two specific mutants of DRnm23 (H134Q and S136P), at residues required for the NDPK activity, inhibit differentiation and promote apoptosis of 32Dcl3 cells. By contrast, substitution of serine 61 with proline (S61P) or deletion of the RGD domain (DeltaRGD) abrogates the effects of wild-type DRnm23. Like wild-type DRnm23, all four mutants show a predominantly mitochondrial subcellular localization. These studies indicate that the enzymatic activity of DRnm23 is not required for the effects observed in 32Dcl3 cells. Moreover, the inability of the S61P and DeltaRGD DRnm23 mutants to inhibit differentiation and promote apoptosis may be due to defective protein-protein interactions at the mitochondria, the predominant site of DRnm23 subcellular localization.

Lee HY, Lee H
Inhibitory activity of nm23-H1 on invasion and colonization of human prostate carcinoma cells is not mediated by its NDP kinase activity.
Cancer Lett. 1999; 145: 93-9
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The human nm23-H1 gene product, a putative metastasis suppressor, was identified as nucleoside diphosphate kinase (NDPK) A isoform. To investigate the functional effect of nm23-H1's NDPK activity on its suppression of the components of metastatic phenotype, we transfected a human prostate carcinoma cell line, DU145, with the cDNA encoding nm23-H1 mutant protein lacking NDPK activity. The mutant nm23-H1 transfected cell lines displayed decreased invasiveness and colonization in soft agar as the wild-type nm23-H1 transfectants did when compared with the control transfected line. The results suggest that the metastasis suppressing function of nm23-H1 is independent of the NDPK enzymatic activity.

Mehus JG, Deloukas P, Lambeth DO
NME6: a new member of the nm23/nucleoside diphosphate kinase gene family located on human chromosome 3p21.3.
Hum Genet. 1999; 104: 454-9
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The NME (nm23/nucleoside diphosphate kinase) gene family in human is involved in the phosphorylation of nucleoside diphosphates and a variety of regulatory phenomena associated with development, oncogenic transformation, and metastasis. Here we report the cDNA sequence for a sixth member of this family, NME6. The cDNA sequence predicts a 186-residue protein that includes the characteristic active site motif of a nucleoside diphosphate (NDP) kinase, as well as the other residues previously identified as crucial for nucleotide binding and catalysis. The NME6 protein sequence is only 34-41% identical to the five previously reported human NME proteins, and is similarly related to prokaryotic and primitive eukaryotic NDP kinases. Compared to typical proteins of this family such as NME1 and NME2, NME6 has three additional residues located in the Kpn loop, and a 22-residue extension at the COOH-terminal. Using radiation hybrid mapping, the NME6 gene was localized to chromosome 3p21.3. The 1.3-kb transcript of NME6 is expressed at a moderately low level in many human tissues, and is most abundant in kidney, prostate, ovary, intestine, and spleen. Homologous cDNAs were also cloned and sequenced for rat and mouse. The sequence of the first 171 residues of the mouse homologue (Nm23-M6) is 94% identical to the deduced human NME6 protein.

Barthel TK, Walker GC
Inferences concerning the ATPase properties of DnaK and other HSP70s are affected by the ADP kinase activity of copurifying nucleoside-diphosphate kinase.
J Biol Chem. 1999; 274: 36670-8
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Preparations of Escherichia coli DnaK from our lab as well as preparations of DnaK and other HSP70 proteins from several major labs in the field produce a stoichiometric initial burst of [alpha-(32)P]ADP when incubated with [alpha-(32)P]ATP and contain an ADP kinase activity. We determined that the initial burst activity results from the transfer of gamma-phosphate from the radiolabeled substrate [alpha-(32)P]ATP to unlabeled ADP bound by the DnaK and is the same activity that results in ADP phosphorylation. The purification of DnaK from E. coli cells that carry a disrupted ndk gene, ndk::km, results in preparations with greatly reduced ADP kinase activities compared with preparations of DnaK purified from ndk(+) cells. The reduction in the amount of ADP kinase activity in preparations of DnaK purified from ndk::km cells shows that nucleoside-diphosphate kinase (NDP kinase) is responsible for most of the ADP kinase activity present in DnaK preparations isolated from ndk(+) cells. The remaining ADP kinase activity in preparations from ndk::km cells, which varies between preparations, is also a property of NDP kinase, which is most likely expressed because of a low frequency reversion of the disrupted ndk gene. A weak, but measurable physical interaction exists between DnaK and NDP kinase and may be at least partially responsible for the co-purification of NDP kinase with DnaK. The presence of contaminating NDP kinase can explain the range of k(cat) values reported for the ATPase activity of DnaK as well as recent reports of initial burst kinetics by DnaK (Banecki, B., and Zylicz, M. (1996) J. Biol. Chem. 271, 6137-6143) and an ADP-ATP exchange activity of DnaK (Hiromura, M., Yano, M., Mori, H., Inoue, M., and Kido, H. (1998) J. Biol. Chem. 273, 5435-5438).

Struglics A, Hakansson G
Purification of a serine and histidine phosphorylated mitochondrial nucleoside diphosphate kinase from Pisum sativum.
Eur J Biochem. 1999; 262: 765-73
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For the first time, to our knowledge, a nucleoside diphosphate kinase (NDPK) has been purified from plant mitochondria (Pisum sativum L.). In intact pea leaf mitochondria, a 17.4-kDa soluble protein was phosphorylated in the presence of EDTA when [gamma-32P]ATP was used as the phosphate donor. Cell fractionation demonstrated that the 17.4-kDa protein is a true mitochondrial protein, and the lack of accessibility to EDTA of the matrix compartment in intact mitochondria suggested it may have an intermembrane space localization. The 17.4-kDa protein was purified from mitochondrial soluble proteins using ATP-agarose and anion exchange chromatography. Amino-acid sequencing of two peptides, resulting from a trypsin digestion, revealed high similarity with the conserved catalytic phosphohistidine site and with the C-terminal of NDPKs. Acid and alkali treatments of [32P]-labelled pea mitochondrial NDPK indicated the presence of acid-stable as well as alkali-stable phosphogroups. Thin-layer chromatography experiments revealed serine as the acid-stable phosphogroup. The alkali-stable labelling probably reflects phosphorylation of the conserved catalytic histidine residue. In phosphorylation experiments, the purified pea mitochondrial NDPK was labelled more heavily on serine than histidine residues. Furthermore, kinetic studies showed a faster phosphorylation rate for serine compared to histidine. Both ATP and GTP could be used as phosphate donor for histidine as well as serine labelling of the pea mitochondrial NDPK.

Admiraal SJ et al.
Nucleophilic activation by positioning in phosphoryl transfer catalyzed by nucleoside diphosphate kinase.
Biochemistry. 1999; 38: 4701-11
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The nonenzymatic reaction of ATP with a nucleophile to generate ADP and a phosphorylated product proceeds via a dissociative transition state with little bond formation to the nucleophile. Consideration of the dissociative nature of the nonenzymatic transition state leads to the following question: To what extent can the nucleophile be activated in enzymatic phosphoryl transfer? We have addressed this question for the NDP kinase reaction. A mutant form of the enzyme lacking the nucleophilic histidine (H122G) can be chemically rescued for ATP attack by imidazole or other exogenous small nucleophiles. The ATP reaction is 50-fold faster with the wild-type enzyme, which has an imidazole nucleophile positioned for reaction by a covalent bond, than with H122G, which employs a noncovalently bound imidazole nucleophile [(kcat/KM)ATP]. Further, a 4-fold advantage for imidazole positioned in the nucleophile binding pocket created by the mutation is suggested from comparison of the reaction of H122G and ATP with an imidazole versus a water nucleophile, after correction for the intrinsic reactivities of imidazole and water toward ATP in solution. X-ray structural analysis shows no detectable rearrangement of the residues surrounding His 122 upon mutation to Gly 122. The overall rate effect of approximately 10(2)-fold for the covalent imidazole nucleophile relative to water is therefore attributed to positioning of the nucleophile with respect to the reactive phosphoryl group. This is underscored by the more deleterious effect of replacing ATP with AlphaTauPgammaS in the wild-type reaction than in the imidazole-rescued mutant reaction, as follows. For the wild-type, AlphaTauPgammaS presumably disrupts positioning between nucleophile and substrate, resulting in a large thio effect of 300-fold, whereas precise alignment is already disrupted in the mutant because there is no covalent bond to the nucleophile, resulting in a smaller thio effect of 10-fold. In summary, the results suggest a catalytic role for activation of the nucleophile by positioning in phosphoryl transfer catalyzed by NDP kinase.

Ogura Y, Yoshida Y, Ichimura K, Aoyagi C, Yabe N, Hasunuma K
Isolation and characterization of Neurospora crassa nucleoside diphosphate kinase NDK-1.
Eur J Biochem. 1999; 266: 709-14
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We have previously reported that phosphorylation of a 15-kDa protein increased after blue-light irradiation in Neurospora crassa. In this study, the 15-kDa protein was purified using four columns; DEAE-cellulose, Blue-Sepharose, SP-Sepharose and Mono Q. The 15-kDa protein was shown to be homologous with nucleoside diphosphate kinase by amino acid sequencing and was also shown to possess nucleoside diphosphate kinase activity. A gene encoding N. crassa nucleoside diphosphate kinase, ndk-1, was isolated from the mycelial cDNA and genomic libraries. The deduced amino acid sequence of NDK-1 was identical to that of the 15-kDa protein. Northern blot analysis suggested that WC-1 and WC-2, the key factors of blue-light signal transduction in N. crassa, did not regulate NDK-1 at the transcriptional level. NDK-1 also showed rapid autophosphorylation activity and protein kinase activity against myelin basic protein with a Km value of 0.36 mM. These results suggest that NDK-1 acts as a signal transducer by phosphorylating proteins.

Pinon VP et al.
Cytoskeletal association of the A and B nucleoside diphosphate kinases of interphasic but not mitotic human carcinoma cell lines: specific nuclear localization of the B subunit.
Exp Cell Res. 1999; 246: 355-67
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The human A and B subunits of nucleoside diphosphate kinase (NDP kinase), encoded by the nm23-H1 and nm23-H2 genes, respectively, associate as homo- or heterohexamers to be catalytically active for the synthesis of nucleoside triphosphates. Despite 88% identity, they appear to possess specific functions. The nm23-H1 gene is implicated in tumor progression and metastasis, and the nm23-H2 gene product is a transcription factor for c-myc. To determine if these distinct functions reflect different subcellular localizations, the distribution of the A and B NDP kinases was analyzed by immunocytofluorescence microscopy in human breast cancer cell lines (MCF-7 and MDA-MB-231) using highly specific polyclonal and monoclonal antibodies. Interphasic cells exhibited a granular and filamentous cytoplasmic staining, particularly intense around nuclei, with both anti-NDP kinase A and B antibodies. The filamentous component observed with either anti-A or anti-B antibodies was altered in parallel to tubulin labeling with compounds interacting with microtubules, such as taxol and colchicine. Confirming published biochemical data, a partial colocalization with the vimentin network was observed in the MDA-231 cell line. A nuclear and nucleolar localization of NDP kinase B was shown by confocal microscopy which was not observed with the A enzyme. In dividing cells, NDP kinase labeling was punctiform and was not colocalized with the mitotic spindle. In conclusion, the A and B NDP kinases are similarly distributed in cytosol, associated partly to microtubules supporting a role in nucleotide channeling. Only the B enzyme is present in nuclei in accord with its role as a DNA binding protein. Their altered localization in dividing cells suggests colocalization with yet unidentified structures which are not intermediate filament aggregates.

Zhu J et al.
Interaction of the Ras-related protein associated with diabetes rad and the putative tumor metastasis suppressor NM23 provides a novel mechanism of GTPase regulation.
Proc Natl Acad Sci U S A. 1999; 96: 14911-8
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Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP.Rad was also reconverted to GTP.Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation.

Gonin P et al.
Catalytic mechanism of nucleoside diphosphate kinase investigated using nucleotide analogues, viscosity effects, and X-ray crystallography.
Biochemistry. 1999; 38: 7265-72
Display abstract
Nucleoside diphosphate (NDP) kinases display low specificity with respect to the base moiety of the nucleotides and to the 2'-position of the ribose, but the 3'-hydroxyl is found to be important for catalysis. We report in this paper the enzymatic analysis of a series of derivatives of thymidine diphosphate (TDP) where the 3'-OH group was removed or replaced by fluorine, azido, and amino groups. With Dictyostelium NDP kinase, kcat decreases 15-200-fold from 1100 s-1 with TDP, and (kcat/Km)NDP decreases from 12 x 10(6) to 10(3) to 5 x 10(4) M-1 s-1, depending on the substrate. The poorest substrates are 3'-deoxyTDP and 3'-azido-3'-deoxyTDP, while the best modified substrates are 2',3'-dehydro-3'-deoxyTDP and 3'-fluoro-3'-deoxyTDP. In a similar way, 3'-fluoro-2',3'-dideoxyUDP was found to be a better substrate than 2',3'-dideoxyUDP, but a much poorer substrate than 2'-deoxyUDP. (kcat/Km)NDP is sensitive to the viscosity of the solution with TDP as the substrate but not with the modified substrates. To understand the poor catalytic efficiency of the modified nucleotides at a structural level, we determined the crystal structure of Dictyostelium NDP kinase complexed to 3'-fluoro-2',3'-dideoxyUDP at 2.7 A resolution. Significant differences are noted as compared to the TDP complex. Substrate-assisted catalysis by the 3'-OH, which is effective in the NDP kinase reaction, cannot occur with the modified substrate. With TDP, the beta-phosphate, which is the leaving group when a gamma-phosphate is transferred to His122, hydrogen bonds to the 3'-hydroxyl group of the sugar; with 3'-fluoro-2',3'-dideoxyUDP, the beta-phosphate hydrogen bonds to Asn119 and moves away from the attacking Ndelta of the catalytic His122. Since all anti-AIDS nucleoside drugs are modified at the 3'-position, these results are relevant to the role of NDP kinase in their cellular metabolism.

Karaschuk G, Kakuev D
Isolation of cDNA clones encoding two isoforms of nucleoside diphosphate kinase from bovine retina.
FEBS Lett. 1999; 449: 83-7
Display abstract
The cDNA encoding bovine retinal isoforms of nucleoside diphosphate kinase (NDP-kinase, EC 2.7.4.6) has been cloned and sequenced. Based on the partial amino acid sequence of the enzyme determined after trypsin digestion of purified NDP-kinase, primers were synthesized and used to isolate two different cDNA clones encoding the full length of two NDP-kinase isoforms. The nucleotide sequences of these clones contained open reading frames encoding 152-residue polypeptides with calculated molecular masses of 17.262 and 17.299 kDa, similar to that determined for the subunits of purified enzyme (17.5 and 18.5 kDa). The deduced NDP-kinase sequences showed high similarity with the known NDP-kinase sequences from other sources.

Tsuiki H et al.
A novel human nucleoside diphosphate (NDP) kinase, Nm23-H6, localizes in mitochondria and affects cytokinesis.
J Cell Biochem. 1999; 76: 254-69
Display abstract
Nucleoside diphosphate kinases (NDP kinases) are enzymes known to be conserved throughout evolution and have been shown to be involved in various biological events, in addition to the "housekeeping" phosphotransferase activity. We present the molecular cloning of a novel human NDP kinase gene, termed Nm23-H6. Nm23-H6 gene has been mapped at chromosome 3p21.3 and is highly expressed in heart, placenta, skeletal muscle, and some of the cancer cell lines. Recombinant Nm23-H6 protein has been identified to exhibit functional NDP kinase activity. Immunolocalization studies showed that both endogenous and inducibly expressed Nm23-H6 proteins were present as short, filament-like, perinuclear radical arrays and that they colocalized with mitochondria. Cell fractionation study also demonstrated the presence of Nm23-H6 protein in a mitochondria-rich fraction. Moreover, induction of overexpression of Nm23-H6 in SAOS2 cells, using the Cre-loxP gene activation system, resulted in growth suppression and generation of multinucleated cells. Flow cytometric analysis also demonstrated that the proportion of cells with more than 4N DNA content increased to 28.1% after induction of Nm23-H6, coinciding with the appearance of multinucleated cells. These observations suggest that Nm23-H6, a new member of the NDP kinase family, resides in mitochondria and plays a role in regulation of cell growth and cell cycle progression.

Postel EH
Cleavage of DNA by human NM23-H2/nucleoside diphosphate kinase involves formation of a covalent protein-DNA complex.
J Biol Chem. 1999; 274: 22821-9
Display abstract
The NM23 gene family in humans is implicated in differentiation and cancer, but the biochemical mechanisms are unknown. Most NM23 proteins have phosphotransferase (nucleoside diphosphate kinase) activity, and the second human isoform, NM23-H2, also binds to a nuclease-hypersensitive c-MYC promoter element through which it activates c-MYC transcription. It is shown here that this DNA binding can result in double-stranded breaks. The DNA breaks occur within repeated sequence elements in the linear nuclease-hypersensitive duplex and leave staggered ends with 5-nucleotide-long 3'-extensions. The enzyme also cleaves supercoiled plasmid DNA to yield nicked circular and unit length linear products. The cleavage reaction requires only NM23-H2, DNA, Mg(2+), and buffer, occurs in the absence of denaturing conditions, and can be reversed by EDTA. The cleaved DNA strands have free 3'-OH groups, and protein is attached to the 5'-phosphoryl ends. Transfer of (32)P radioactivity from DNA to NM23-H2 has been observed, and a covalent polypeptide-DNA complex has been isolated and identified by Western blotting as NM23-H2. Since covalent protein-DNA complexes are known to serve the role of breaking and rejoining DNA strands, the present findings suggest that NM23-H2 is involved in DNA structural transactions necessary for the activity of the c-MYC promoter.

Schneider B et al.
Phosphorylation of anti-HIV nucleoside analogs by nucleoside diphosphate kinase.
Nucleosides Nucleotides. 1999; 18: 829-30
Display abstract
The reaction of NDP kinase with antiviral nucleoside triphosphates used in antiviral therapies was studied at the presteady state by fluorescence stopped-flow and compared with the steady-state parameters. The affinity of the analogs was determined by fluorescence titration of a mutated enzyme with an inserted Trp in the binding site. The lack of the 3' hydroxyl in analogs is shown to decrease the kcat more than the KD.

Prinz H, Lavie A, Scheidig AJ, Spangenberg O, Konrad M
Binding of nucleotides to guanylate kinase, p21(ras), and nucleoside-diphosphate kinase studied by nano-electrospray mass spectrometry.
J Biol Chem. 1999; 274: 35337-42
Display abstract
The binding of nucleotides to three different nucleotide-binding proteins and to a control protein was studied by means of nano-electrospray mass spectrometry applied to aqueous nondenaturing solutions. The method leads to unambiguous identification of enzyme complexes with substrates and products but does not allow the determination of dissociation constants or even stoichiometries relevant to the binding in solution. For guanylate kinase (EC 2.7.4. 8), the transfer of HPO(3) between nucleotides was observed whenever a ternary complex with adenylate or guanylate nucleotides was formed. Guanosine 5'-tetraphosphate was generated after prolonged incubation with GDP or GTP. Mg(2+) binding was considerably enhanced in functional high affinity complexes, such as observed between guanylate kinase and its bisubstrate inhibitor P(1)-(5'-guanosyl)-P(5)-(5'-adenosyl) pentaphosphate or with the tight nucleotide-binding protein p21(ras) and GDP. Nucleoside-diphosphate kinase (EC 2.7.4.6) itself was phosphorylated in accordance to its known ping-pong mechanism. All nucleotide-binding proteins were shown to bind sulfate (SO(4)(2-)) with presumably high affinity and slow exchange rate. The binding of phosphate (PO(4)(3-)) could be inferred indirectly from competition with SO(4)(2-).

Karashchuk GN, Kakuev DL, Popov VI, Gaidarov IO, Abdulaev NG
[Biochemical characteristics of bovine retina nucleoside diphosphate kinase]
Bioorg Khim. 1999; 25: 513-9
Display abstract
Nucleoside diphosphate kinase (NDP kinase; ATP: NDP phosphotransferase; EC 2.7.4.6) was purified from bovine retina. The molecular mass of the native enzyme was found to be 72 kDa, and those of its subunits were 17.5 and 18.5 kDa. Kinetic characteristics of the enzyme were determined. It was shown that NDP kinase exists in retina in both soluble and membrane-bound forms.

Dabernat S et al.
Cloning of a second nm23-M1 cDNA: expression in the central nervous system of adult mouse and comparison with nm23-M2 mRNA distribution.
Brain Res Mol Brain Res. 1999; 63: 351-65
Display abstract
Nm23 has been identified as a gene family encoding different isoforms of the nucleoside diphosphate kinase. This protein is a key enzyme in the control of cellular concentrations of nucleoside triphosphates. Moreover, it has been shown to play important roles in various cellular functions such as differentiation and metastasis. In the present study, a second cDNA for nucleoside diphosphate kinase A (Nm23-M1) was isolated from a cDNA library of mouse embryonic stem cells. This clone encodes the same putative 152 aminoacids long protein as an already published cDNA but is longer in both its 5' and 3' untranslated regions. Tissue and cellular distribution of nm23-M1 mRNA was investigated by using Northern blot analysis and in situ hybridization. Nm23-M1 transcripts were found to be widely distributed throughout the mouse central nervous system with prominent expression in several restricted areas. No differences were noticed between the distribution of long and short transcripts. Furthermore, a similar pattern of expression was described in the central nervous system for nm23-M2 mRNA, encoding a second isoform of the nucleoside diphosphate kinase. However, the transcript of this isoform displayed a wider distribution and was expressed in all organs analysed by northern blotting. The possible involvement of nm23-M1 in differentiation of mouse nervous system is further discussed.

Karamohamed S, Nordstrom T, Nyren P
Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity.
Biotechniques. 1999; 26: 728-34
Display abstract
A real-time, simple and sensitive method for detection of nucleoside diphosphate (NDP) kinase activity has been developed. The assay is based on detection of ATP, generated in the NDP kinase reaction between a nucleoside triphosphate and adenosine diphosphate (ADP), by the firefly luciferase system. In the presence of 0.3 mM dGTP, the Km for ADP was found to be approximately 30 microM for the NDP kinase from Baker's yeast. In the presence of 250 microM ADP, the Km for dATP alpha S, dTTP alpha S, dGTP, dTTP, dCTP and GTP was found to be approximately 0.01, 0.03, 0.05, 0.25, 0.75 and 0.2 mM, respectively. The assay is sensitive and yields linear responses between 0.05-50 mU. The detection limit was found to be 0.05 mU of NDP kinase. The method was used to detect NDP kinase contamination in commercial enzyme preparations.

Marshall LJ, Muimo R, Riemen CE, Mehta A
Na+ and K+ regulate the phosphorylation state of nucleoside diphosphate kinase in human airway epithelium.
Am J Physiol. 1999; 276: 10919-10919
Display abstract
We describe how cations, in the presence of ATP, regulate the phosphorylated form of 19- and 21-kDa nucleoside diphosphate kinase (NDPK; EC 2.7.4.6), a kinase controlling K+ channels, G proteins, cell secretion, cellular energy production, and UTP synthesis. In apically enriched human nasal epithelial membranes, 10 mM Na+ inhibits phosphorylation of NDPK relative to other cations. Dose response showed that, whereas K+ induces a fourfold greater phosphate incorporation (EC50 10 mM), Na+ is inhibitory (EC50 10 mM) compared with respective buffer controls. Cation discrimination is nucleotide selective (not seen with [gamma-32P]GTP) and NDPK specific (not seen with p37h, a previously characterized Cl--sensitive phosphoprotein). Na+ does not exert an inhibitory effect on NDPK phosphorylation directly but is likely to act via an okadaic acid-insensitive phosphatase. We speculate that the ability of NDPK to discriminate between physiologically relevant cation concentrations provides a novel example of cross talk within the apical membrane.

Yano M, Mori S, Kido H
Intrinsic nucleoside diphosphate kinase-like activity is a novel function of the 20 S proteasome.
J Biol Chem. 1999; 274: 34375-82
Display abstract
The eukaryotic 20 S proteasome is the prototype of a new family of the N-terminal nucleophil hydrolases and is composed of numerous low molecular mass subunits arranged in a stack of four rings, each containing seven different alpha- or beta-subunits. Among the beta-type subunits in the yeast proteasome, three proteolytically active ones were identified, although the functions of the other beta- and alpha-type subunits remain to be clarified. We report here that the purified 20 S proteasome exhibits intrinsic nucleoside diphosphate (NDP) kinase-like activity. The proteasome exhibited a preference for ATP and dATP as phosphate donors, and a broad specificity for NDPs, other than GDP, as phosphate acceptors, unlike conventional NDP kinase, which catalyzes the transfer of gamma-phosphate between NDPs and nucleoside triphosphates. During the transfer of gamma-phosphate, the proteasome formed acid-labile phosphohistidine as autophosphorylated intermediates, and NDP-dependent dephosphorylation of the latter then occurred. These enzymatic properties are similar to those of the molecular chaperone, Hsp70, which also exhibits intrinsic NDP kinase-like activity, instead of ATPase activity. C5 among the beta-type subunits and C8 among the alpha-type subunits were autophosphorylated during the gamma-phosphate transfer reaction and were photoaffinity labeled with 8-azido-[alpha-(32)P]ATP, suggesting that the C5 and C8 subunits of the proteasome are responsible for the NDP kinase-like activity.

Abdulaev NG et al.
Nucleoside diphosphate kinase from bovine retina: purification, subcellular localization, molecular cloning, and three-dimensional structure.
Biochemistry. 1998; 37: 13958-67
Display abstract
The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated. The enzyme showed two polypeptides of approximately 17.5 and 18.5 kDa on SDS-PAGE, while isoelectric focusing revealed seven to eight proteins with a pI range of 7.4-8.2. Sedimentation equilibrium yielded a molecular mass of 96 +/- 2 kDa for the enzyme. Carbohydrate analysis revealed that both polypeptides contained Gal, Man, GlcNAc, Fuc, and GalNac saccharides. Like other nucleoside diphosphate kinases, the retinal enzyme showed substantial differences in the Km values for various di- and triphosphate nucleotides. Immunogold labeling of bovine retina revealed that the enzyme is localized on both the membranes and in the cytoplasm. Screening of a retinal cDNA library yielded full-length clones encoding two distinct isoforms (NBR-A and NBR-B). Both isoforms were overexpressed in Escherichia coli and their biochemical properties compared with retinal NDP-kinase. The structures of NBR-A and NBR-B were determined by X-ray crystallography in the presence of guanine nucleotide(s). Both isoforms are hexameric, and the fold of the monomer is similar to other nucleoside diphosphate kinase structures. The NBR-A active site contained both a cGMP and a GDP molecule each bound at half occupancy while the NBR-B active site contained only cGMP.

Ouatas T, Selo M, Sadji Z, Hourdry J, Denis H, Mazabraud A
Differential expression of nucleoside diphosphate kinases (NDPK/NM23) during Xenopus early development.
Int J Dev Biol. 1998; 42: 43-52
Display abstract
In Xenopus laevis, three nucleoside diphosphate kinase (NDPK) monomers have been described (NDPK X1, X2 and X3) (Ouatas et al., 1997). In eucaryotes, this kinase is known as a hetero- or homohexamer. Here, we examine the distribution of the enzyme and its different subunit mRNAs during oogenesis and early embryogenesis of Xenopus laevis, respectively by immunohistofluorescence and whole-mount in situ hybridization. These analyses show that NDPKs and their mRNAs are differentially distributed throughout the oocyte and early embryos with a high level of transcription in somites and brain. We emphasize two points. First, each mRNA displays a distinct subcellular localization in somites, suggesting a complex regulation of NDPK genes both at the transcriptional and translational level and a possible involvement of NDPK X2 homohexamers in the dorsal muscle differentiation. Second, in oocytes and early embryos, the proteins are mainly localized in the nucleus, suggesting a new mechanism for their nuclear import, since they do not possess any known nuclear import sequences.

Mesnildrey S, Agou F, Karlsson A, Bonne DD, Veron M
Coupling between catalysis and oligomeric structure in nucleoside diphosphate kinase.
J Biol Chem. 1998; 273: 4436-42
Display abstract
A dimeric Dictyostelium nucleoside diphosphate kinase has been stabilized by the double mutation P100S-N150stop which targets residues involved in the trimer interface (Karlsson, A., Mesnildrey, S., Xu, Y., Morera, S., Janin, J., and Veron, M. (1996) J. Biol. Chem. 271, 19928-19934). The reassociation of this dimeric form into a hexamer similar to the wild-type enzyme is induced by the presence of a nucleotide substrate. Equilibrium sedimentation and gel filtration experiments, as well as enzymatic activity measurements, show that reactivation of the enzyme closely parallels its reassociation. A phosphorylatable intermediate with low activity participates in the association pathway while the dimeric form is shown totally devoid of enzymatic activity. Our results support the hypothesis that different oligomeric species of nucleoside diphosphate kinase are involved in different cellular processes where the enzymatic activity is not required.

Polosina YYu, Jarrell KF, Fedorov OV, Kostyukova AS
Nucleoside diphosphate kinase from haloalkaliphilic archaeon Natronobacterium magadii: purification and characterization.
Extremophiles. 1998; 2: 333-8
Display abstract
An ATP-binding protein from the haloalkaliphilic archaeon Natronobacterium magadii was purified and characterized by affinity chromatography on ATP-agarose and by fast protein liquid chromatography (FPLC) on a Mono Q column. The N-terminal 20 amino acid sequence of the kinase showed a strong sequence similarity of this protein with nucleoside diphosphate (NDP) kinases from different organisms and, accordingly, we believe that this protein is a nucleoside diphosphate kinase, an enzyme whose main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates. Comparison of the molecular weights of the NDP kinase monomer determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (23,000) and of the oligomer determined by sedimentation equilibrium experiments (125,000) indicated that the oligomer is a hexamer. The enzyme was autophosphorylated in the presence of [gamma-32P]ATP, and Mg2+ was required for the incorporation of phosphate. The kinase preserved the ability to transfer gamma-phosphate from ATP to GDP in the range of NaCl concentration from 90 mM to 3.5 M and in the range of pH from 5 to 12. It was found and confirmed by Western blotting that this kinase is one of the proteins that bind specifically to natronobacterial flagellins. NDP kinase from haloalkaliphiles appeared to be simple to purify and to be a suitable enzyme for studies of structure and stability compared with NDP kinases from mesophilic organisms.

Nosaka K, Kawahara M, Masuda M, Satomi Y, Nishino H
Association of nucleoside diphosphate kinase nm23-H2 with human telomeres.
Biochem Biophys Res Commun. 1998; 243: 342-8
Display abstract
Telomeres, the ends of eukaryotic chromosomes, are essential structures formed by specific protein-DNA complexes that protect chromosomes from degradation and end-to-end fusion. TRF1, a double-stranded telomeric TTAGGG-repeat binding protein, is associated with mammalian telomeres and controls telomere length by inhibiting the action of telomerase. We identified human nucleoside diphosphate kinase nm23-H2 as a human TRF1-interacting protein by yeast two-hybrid screening. In vitro-binding assays using different recombinant nucleoside diphosphate kinases showed that TRF1 predominantly binds the nm23-H2 isoform rather than nm23-H1. Electrophoretic mobility shift analysis revealed that the recombinant nm23-H2 protein can bind the single-stranded telomeric TTAGGG-repeat while it cannot bind the double-stranded telomeric repeat. The synthetic 20 base oligoribonucleotide, which consists of the template sequence CUAACCCUAAC and the adjacent region of the RNA component of human telomerase, was also found to form the complex with the recombinant nm23-H2 protein. Furthermore, the affinity of telomerase for its substrate was increased in vitro by presence of the plentiful nm23-H2 protein. These findings indicate a close relationship between nm23-H2 and human telomeres and suggest a new biological role for nucleoside diphosphate kinase.

Deville-Bonne D, Schneider B, Bourdais J, Veron M
Reaction of 2',3'-dideoxynucleotides triphosphates with recombinant human nucleoside diphosphate kinase.
Adv Exp Med Biol. 1998; 431: 569-73

Tagashira H, Hamazaki K, Tanaka N, Gao C, Namba M
Reduced metastatic potential and c-myc overexpression of colon adenocarcinoma cells (Colon 26 line) transfected with nm23-R2/rat nucleoside diphosphate kinase alpha isoform.
Int J Mol Med. 1998; 2: 65-8
Display abstract
Increased expression of nm23/nucleoside diphosphate kinase (NDP kinase) has been reported to be associated with both reduced metastatic potential in breast carcinoma and tumor progression in colon adenocarcinoma and lung adenocarcinoma. We examined effects of expression of nm23-R2 rat NDP kinase alpha isoform on mouse adenocarcinoma cells (Colon 26 line) and found a significant reduction of metastatic potential along with overexpression of c-myc. We also found that the proliferation rate of the transformed cells was the same as that of the control cells in culture. These results indicate that the cell growth potential in vitro is irrelevant to metastatic potential of the cells in vivo.

Willems R et al.
Decrease in nucleoside diphosphate kinase (NDPK/nm23) expression during hematopoietic maturation.
J Biol Chem. 1998; 273: 13663-8
Display abstract
The nucleoside diphosphate kinase (NDPK/nm23) isoforms H1 and H2 were localized in hematopoietic tissues. Flow cytometric analysis and enzymatic assays were used to quantify the intracellular and extracellular concentrations of NDPK. Bone marrow CD34(+) progenitors contained the highest intracellular levels of both nm23-H1 and nm23-H2. Lower levels were measured in more mature bone marrow cells, whereas peripheral blood leukocytes had the lowest expression of nm23. These data suggest a function of NDPK in early hematopoiesis and a down-regulation of NDPK upon differentiation. In addition, an up-regulation of nm23 expression was observed in lymphocytes after induction of proliferation with phytohemagglutinin. Multiparameter flow cytometry demonstrated that this up-regulation occurred during the G0/G1-transition. Flow cytometric analysis also revealed a weak surface expression of nm23 on a number of hematopoietic cell lines, which was not detected on normal hematopoietic cells. Our data also demonstrated the presence of NDPK in human plasma, probably due to a limited in vivo lysis of red blood cells.

Schneider B, Xu YW, Janin J, Veron M, Deville-Bonne D
3'-Phosphorylated nucleotides are tight binding inhibitors of nucleoside diphosphate kinase activity.
J Biol Chem. 1998; 273: 28773-8
Display abstract
Nucleoside diphosphate (NDP) kinase catalyzes the phosphorylation of ribo- and deoxyribonucleosides diphosphates into triphosphates. NDP kinase is also involved in malignant tumors and was shown to activate in vitro transcription of the c-myc oncogene by binding to its NHE sequence. The structure of the complex of NDP kinase with bound ADP shows that the nucleotide adopts a different conformation from that observed in other phosphokinases with an internal H bond between the 3'-OH and the beta-O made free by the phosphate transfer. We use intrinsic protein fluorescence to investigate the inhibitory and binding potential of nucleotide analogues phosphorylated in 3'-OH position of the ribose to both wild type and F64W mutant NDP kinase from Dictyostelium discoideum. Due to their 3'-phosphate, 5'-phosphoadenosine 3'-phosphate (PAP) and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) can be regarded as structural analogues of enzyme-bound ADP. The KD of PAPS (10 microM) is three times lower than the KD of ADP. PAPS also acts as a competitive inhibitor toward natural substrates during catalysis, with a KI in agreement with binding data. The crystal structure of the binary complex between Dictyostelium NDP kinase and PAPS was solved at 2.8-A resolution. It shows a new mode of nucleotide binding at the active site with the 3'-phosphate of PAPS located near the catalytic histidine, at the same position as the gamma-phosphate in the transition state. The sulfate group is directed toward the protein surface. PAPS will be useful for the design of high affinity drugs targeted to NDP kinases.

Schaertl S, Konrad M, Geeves MA
Substrate specificity of human nucleoside-diphosphate kinase revealed by transient kinetic analysis.
J Biol Chem. 1998; 273: 5662-9
Display abstract
Nucleoside-diphosphate kinases (NDKs) catalyze the transfer of gamma-phosphoryl groups from NTPs via an active site histidine to NDPs using a ping-pong mechanism. We have used the change of intrinsic tryptophan fluorescence that occurs upon phosphorylation of NDK to measure the rates of phosphorylation and dephosphorylation with a range of nucleotides and nucleotide analogues. For natural nucleotides, the rates of phosphorylation and dephosphorylation were linearly dependent upon nucleotide concentration until they became too fast to measure. The second order rate constants for phosphorylation by natural NTPs varied between 0.7 and 13 x 10(6) M-1 s-1. Dephosphorylation by NDPs was 2-3-fold faster than the corresponding phosphorylation reaction, and dephosphorylation by dNDPs was 3-4-fold slower than the equivalent NDPs. In all cases, second order rate constants were highest for guanine followed by adenine and lowest for cytosine nucleotides. NDK also catalyzes the transfer of thiophosphate from adenosine 5'-O-(thiotriphosphate) (ATPgammaS) and guanosine 5'-O-(thiotriphosphate) (GTPgammaS) to NDP, but at (1)/(1000) of the equivalent phosphoryl transfer rates. In this case, the observed rate constants of phosphorylation and dephosphorylation were hyperbolically dependent on nucleotide concentration. Thiophosphorylation by ATPgammaS and GTPgammaS occurred with kmax of 2.8 and 1.35 s-1 and Kd of 145 and 36 muM respectively. For dethiophosphorylation by a range of NDPs, kmax was in the range of 5-30 s-1, whereas Kd varied between 0.16 and 3.3 mM. Guanine had the lowest Kd values, and cytosine had the highest. The data are consistent with fast reversible binding of the nucleotide followed by the rate-limiting phosphoryl transfer. Thiophosphates change only the rate of the phosphoryl transfer step, whereas both events are influenced by the base. Modification at the 2'-hydroxyl of ribose has only a small effect, while the overall rate of phosphoryl transfer is reduced 1000-fold by modification at the 3'-ribose.

Engel M, Seifert M, Theisinger B, Seyfert U, Welter C
Glyceraldehyde-3-phosphate dehydrogenase and Nm23-H1/nucleoside diphosphate kinase A. Two old enzymes combine for the novel Nm23 protein phosphotransferase function.
J Biol Chem. 1998; 273: 20058-65
Display abstract
We have recently discovered an alternative function of the putative metastasis suppressor protein Nm23, which is identical to nucleoside diphosphate kinase, as a protein phosphotransferase in vitro. While purified native Nm23 protein did not phosphorylate other proteins, we could purify a Nm23-associated protein that activates the protein phosphotransferase function; it was identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isoenzyme. Co-expression and purification of (His)6-tagged GAPDH in combination with either Nm23-H1 or Nm23-H2 in baculovirus-infected Sf9 cells showed that only Nm23-H1, but not Nm23-H2, forms a stable complex with GAPDH. Protein phosphotransferase activity was confirmed for the recombinant GAPDH.Nm23-H1 complex but not for either of the enzymes alone, nor was this activity observed after simple mixing of the purified proteins in vitro. The molecular mass of the highly purified recombinant GAPDH.Nm23-H1 complex suggests that a dimer of GAPDH interacts with a dimer of Nm23-H1. In contrast to the complex with GAPDH, co-expression of Nm23-H1 with antioxidant protein (MER-5) or creatine kinase did not activate the protein phosphotransferase function, indicating that this activation may specifically require GAPDH as a binding partner.

Tanaka N, Ogura T, Noguchi T, Hirano H, Yabe N, Hasunuma K
Phytochrome-mediated light signals are transduced to nucleoside diphosphate kinase in Pisum sativum L. cv. Alaska.
J Photochem Photobiol B. 1998; 45: 113-21
Display abstract
To clarify the molecular mechanism for the transduction of light signals in plants, we have established an in vitro system that uses crude membrane and soluble fractions of stem sections of etiolated Pisum sativum L. cv. Alaska after irradiation by red light, or sequential application of red and far-red light to the stem section. In a previous report (T. Hamada et al., J. Photochem. Photobiol. B: Biol. 33 (1996) 143-151) the labelling of proteins in membrane fraction by [gamma-32P] ATP at 0 degree C for 15 s and subsequent separation of proteins by two-dimensional electrophoresis allowed unambiguous identification of a heavily phosphorylated protein spot at 18 kDa (p18). In the present study we have confirmed the former results in the membrane fraction, and obtained the result that an increase in the phosphorylation of p18 by red-light irradiation is observed in the soluble fraction. Further, we have provided evidence that the p18 in the soluble fraction is purified and identified as nucleoside diphosphate (NDP) kinase by Western blotting, immuno-precipitation, amino acid sequencing and cDNA analysis. Purified p18 shows autophosphorylation activity and strong phosphorylating activity against myelin basic protein (MBP), a substrate of MAP (mitogen activated protein) kinase. The results show that phytochrome-mediated light signals are transduced to NDP kinase, which may elicit signals by providing high concentrations of, for example, GTP from GDT and ATP, by the autophosphorylation and by the protein kinase activity similar to MAP kinase.

Okada K, Shiku H
[Molecular biology and multifunctional aspects of nm23/NDPK]
Seikagaku. 1998; 70: 337-46

Biondi RM, Schneider B, Passeron E, Passeron S
Role of Mg2+ in nucleoside diphosphate kinase autophosphorylation.
Arch Biochem Biophys. 1998; 353: 85-92
Display abstract
Nucleoside diphosphate (NDP) kinase is a ubiquitous enzyme that has been described to have regulatory functions. In addition to its classical enzymatic activity, NDP kinases have been characterized as inhibitors of metastasis, as a factor stimulating gene transcription, and as a protein kinase. In this report we show some characteristics of the autophosphorylation of homogeneous NDP kinase and make a comparison with that of other proteins in crude extracts. By using labeled substrates and fluorescence quenching analysis, we prove that Mg2+ is indeed necessary for the two steps of the ping-pong reaction to take place and present evidence that NTPs or NDPs, when uncomplexed to divalent cations, may not bind the active site in a comparable way to NTP . Mg2+ and NDP . Mg2+. However, even extremely small concentrations of Mg2+ suffice for maximal autophosphorylation which is obtained with Mg2+ in the nanomolar range and 100 microM ATP using homogeneous enzyme. Moreover, lower autophosphorylation levels were observed with increasing concentrations of Mg2+. The autophosphorylation equilibrium varied from 0.19 to 1.6 upon the inclusion of 10 mM EDTA to produce low Mg2+ concentrations. Under optimal conditions (low Mg2+ concentrations and short incubation times) NDP kinase was the only protein phosphorylated in crude extracts from Candida albicans, indicating that the autophosphorylation properties of the enzyme are very singular.

Kowluru A, Kowluru RA
Subcellular localization and characterization of nucleoside diphosphate kinase in rat retina: effect of diabetes.
Biosci Rep. 1998; 18: 187-98
Display abstract
Nucleoside diphosphate kinase (NDP kinase) catalyzes the transfer of terminal phosphate from nucleotide triphosphates (e.g. ATP) to nucleotide diphosphates (e.g. GDP) to yield nucleotide triphosphates (e.g. GTP). Since guanine nucleotides play critical role(s) in GTP-binding protein (G-protein)-mediated signal transduction mechanisms in retina, we quantitated NDP kinase activity in subcellular fraction-derived from normal rat retina. A greater than 85% of the total specific activity was present in the soluble fraction, which was stimulated (up to 7 fold) by 2 mM magnesium. NDP kinase exhibited saturation kinetics towards di- and tri-phosphate substrates, and was inhibited by known inhibitors of NDP kinase, uridine diphosphate (UDP) or cromoglycate (CRG). We have previously reported significant abnormalities in the activation of G-proteins in streptozotocin (STZ)-diabetic rat retina (Kowluru et al. Diabetologia 35:624 631, 1992). Since NDP kinase has been implicated in direct interaction with and/or activation of various G-proteins, we quantitated both basal and magnesium-stimulated NDP kinase activity in soluble and particulate fractions of retina derived from STZ-diabetic rats to examine whether abnormalities in G-protein function in diabetes are attributable to alterations in retinal NDP kinase. There was no effect of diabetes either on the basal or the magnesium-activated retinal NDP kinase activity. This study represents the first characterization of NDP kinase activity in rat retina, and suggests that in diabetes, this enzyme may not be rate-limiting and/or causal for the observed alterations in retinal G-protein functions.

Miyazaki H
[Expression and anti-metastatic potential of nm 23/NDP kinase in human head and neck cancer cells]
Kokubyo Gakkai Zasshi. 1998; 65: 189-95
Display abstract
The nm23, encoding a protein identical to nucleoside diphosphate (NDP) kinase, is thought to be a candidate metastasis suppressor gene. Low expression of nm23 at the protein or/and mRNA levels was associated with metastatic potential of several types of rodent and human tumors. Expression and anti-metastatic action of nm23/NDP kinase gene products of human head and neck cancer (squamous cell carcinoma) cell lines were investigated. Among six cell lines examined, only those which showed metastatic potential exhibited reduced levels of nm23-H2 protein with no apparent change in nm23-H1 protein levels as compared with non-metastatic cells. When either nm23-H1 or nm23-H2 cDNA was transfected into a high metastatic cell line, only cell clones expressing higher levels of nm23-H2 protein above control levels displayed a reduced number of lung foci in an experimental metastasis assay. The data suggested that nm23-H2 gene product possesses the metastasis suppressor activity in human head and neck cancer cells.

Hasunuma K, Yabe N
[Early events occurring during light signal transduction in plants]
Tanpakushitsu Kakusan Koso. 1998; 43: 1443-52

Lee IH, Chang SI, Okada K, Baba H, Shiku H
Transcription effect of nm23-M2/NDP kinase on c-myc oncogene.
Mol Cells. 1997; 7: 589-93
Display abstract
Transactivating factor PuF which interacts with a nuclease hypersensitive element locates upstream from the c-myc gene. PuF was recently identified as being encoded by nm23-H2/ NDP kinase gene [Postel, E. H., Berberich, S. J., Flint, S. J., and Ferrone, C. A. (1993) Science 261, 428-429]. Here we have studied the correlation of expression between c-myc and nm23 genes in vitro. By a comparative study of the expression of 2 genes in cell lines, no direct correlation of kinetics was found. A plasmid containing the human c-myc fragment (c-myc CAT) was cloned upstream from the bacterial chloramphenicol acetyltransferase (CAT) gene. When the murine melanoma cell line was cotransfected with a nm23-M2/ NDP kinase expression vector and c-myc CAT, CAT activity was elevated. In addition, cell cycle phases in the murine cell lines transfected with nm23/NDP kinase were estimated; an alteration of the cell cycle, prolonged S-phase was found in the cell lines transfected with nm23-M2/NDP kinase, whereas human nm23-H2/NDP kinase did not play a role in transactivating the c-myc gene or in S-phase prolongation in murine cell lines. From these results we conclude that the murine nm23-M2 gene transactivates the c-myc gene and controls the cell cycle, S-phase, indirectly via a cellular cofactor in the murine cell line, which does not work with the human nm23-H2 gene.

Yano M, Mori S, Niwa Y, Inoue M, Kido H
Intrinsic nucleoside diphosphate kinase-like activity as a novel function of 14-3-3 proteins.
FEBS Lett. 1997; 419: 244-8
Display abstract
14-3-3 proteins play a role in many cellular functions as molecular chaperone and adapter proteins: they bind to and modulate several proteins involved in cell proliferation and differentiation, and also function ATP-dependently in targeting of precursors to mitochondria. We show here that 14-3-3 purified from a human lymphoblastoma and also its recombinant tau isoform exhibited intrinsic nucleoside diphosphate (NDP) kinase-like activity. 14-3-3 proteins preferentially catalyzed the transfer of the gamma-phosphate group from ATP, dATP or dGTP to all nucleoside diphosphates and this transfer involved acid-labile phosphoenzyme intermediates. They also simultaneously catalyzed the reverse reaction of ATP hydrolysis. These properties of 14-3-3 are similar to those of NDP kinase, but not to those of adenylate kinase.

Kuroda A, Kornberg A
Polyphosphate kinase as a nucleoside diphosphate kinase in Escherichia coli and Pseudomonas aeruginosa.
Proc Natl Acad Sci U S A. 1997; 94: 439-42
Display abstract
Generation of a wide variety of nucleoside (and deoxynucleoside) triphosphates (NTPs) from their cognate nucleoside diphosphates (NDPs) is of critical importance in virtually every aspect of cellular life. Their function is fulfilled largely by the ubiquitous and potent nucleoside diphosphate kinase (NDK), most commonly using ATP as the donor. Considerable interest is attached to the consequence to a cell in which the NDK activity becomes deficient or over-abundant. We have discovered an additional and possibly auxiliary NDK-like activity in the capacity of polyphosphate kinase (PPK) to use inorganic polyphosphate as the donor in place of ATP, thereby converting GDP and other NDPs to NTPs. This reaction was observed with the PPK activity present in crude membrane fractions from Escherichia coli and Pseudomonas aeruginosa as well as with the purified PPK from E. coli; the activity was absent from the membrane fractions obtained from E. coli mutants lacking the ppk gene. The order of substrate specificity for PPK was: ADP > GDP > UDP, CDP; activity with ADP was 2-60 times greater than with GDP, depending on the reaction condition. Although the transfer of a phosphate from polyphosphate to GDP by PPK to produce GTP was the predominant reaction, the enzyme also transferred a pyrophosphate group to GDP to form the linear guanosine 5' tetraphosphate.

Wagner PD, Steeg PS, Vu ND
Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity.
Proc Natl Acad Sci U S A. 1997; 94: 9000-5
Display abstract
Nm23 genes, which encode nucleoside diphosphate kinases, have been implicated in suppressing tumor metastasis. The motility of human breast carcinoma cells can be suppressed by transfection with wild-type nm23-H1, but not by transfections with two nm23-H1 mutants, nm23-H1(S12OG) and nm23-H1(P96S). Here we report that nm23-H1 can transfer a phosphate from its catalytic histidine to aspartate or glutamate residues on 43-kDa membrane proteins. One of the 43-kDa membrane proteins was not phosphorylated by either nm23-H1(P96S) or nm23-H1(S120G), and another was phosphorylated much more slowly by nm23-H1(P96S) and by nm23-H1(S120G) than by wild-type nm23-H1. Nm23-H1 also can transfer phosphate from its catalytic histidine to histidines on ATP-citrate lyase and succinic thiokinase. The rates of phosphorylation of ATP-citrate lyase by nm23-H1(S120G) and nm23-H1(P96S) were similar to that by wild-type nm23-H1. The rate of phosphorylation of succinic thiokinase by nm23-H1(S120) was similar to that by wild-type nm23-H1, and the rate of phosphorylation of succinic thiokinase by nm23-H1(P96S) was about half that by wild-type nm23-H1. Thus, the transfer of phosphate from nm23-H1 to aspartates or glutamates on other proteins appears to correlate better with the suppression of motility than does the transfer to histidines.

Royds JA, Robinson MH, Stephenson TJ, Rees RC, Fisher C
The association between nm23 gene expression and survival in patients with sarcomas.
Br J Cancer. 1997; 75: 1195-200
Display abstract
The relationship between the expression of nm23, a putative metastasis-suppressor gene and prognosis was determined for 88 patients with sarcomas. Immunohistochemistry using immunopurified anti-nm23 peptide antibodies was performed and the results of each case graded according to the degree of staining. Univariate and multivariate analyses were carried out to determine the prognostic significance of nm23 staining for sarcoma patients. Expression of nm23 was found to increase in line with metastatic potential in many cases but this did not reach significance for the study as a whole. However, the possibility of nm23 loss occurring in association with metastasis cannot be ruled out in some more aggressive sarcomas, as was demonstrated for six patients with low-scoring, unclassified and synovial sarcomas that had metastasized. The time to metastasis was longer for patients with grade 3 sarcomas (50-75% of tumour cells staining) than similar patients in other staining groups. These results suggest that expression of nm23 genes in sarcomas is variable and has no value as a prognostic indicator for these mesenchymal tumours.

Xu Y, Lecroisey A, Veron M, Delepierre M, Janin J
NMR studies on the flexibility of nucleoside diphosphate kinase.
Proteins. 1997; 28: 150-2
Display abstract
Human NDP kinase B, product of the nm23-H2 gene, binds DNA. It has been suggested that a helix hairpin on the protein surface, part of the nucleotide substrate binding site, could accommodate DNA binding by swinging away. The presence of flexible regions was therefore investigated by 1H NMR dynamic filtering. Although TOCSY peaks could be assigned to five residues at the N terminus of Dictyostelium NDP kinase, no flexible region was detected in the human enzyme. These data favor the idea that the protein offers different binding sites to mono- and polynucleotides.

Milon L et al.
nm23-H4, a new member of the family of human nm23/nucleoside diphosphate kinase genes localised on chromosome 16p13.
Hum Genet. 1997; 99: 550-7
Display abstract
A novel human nm23/nucleoside diphosphate (NDP) kinase gene, called nm23-H4, was identified by screening a human stomach cDNA library with a probe generated by amplification by reverse transcription-polymerase chain reaction. The primers were designed from publicly available database cDNA sequences selected according to their homology to the human nn23-H1 putative metastasis suppressor gene. The full-length cDNA sequence predicts a 187 amino acid protein possessing the region homologous to NDP kinases with all residues crucial for nucleotide binding and catalysis, strongly suggesting that Nm23-H4 possesses NDP kinase activity. It shares 56, 55 and 60% identity with Nm23-H1, Nm23-H2 and DR-Nm23, respectively, the other human Nm23 proteins isolated so far. Compared with these proteins, Nm23-H4 contains an additional NH2-terminal region that is rich in positively charged residues and could indicate routing to mitochondria. The nm23-H4 gene has been localised to human chromosomal band 16p13.3. The corresponding 1.2 kb mRNA is widely distributed and expressed in a tissue-dependent manner, being found at very high levels in prostate, heart, liver, small intestine and skeletal muscle tissues and in low amounts in the brain and in blood leucocytes. Nm23-H4 naturally possesses the Pro-Ser substitution equivalent to the K-pn mutation (P97S) of Drosophila.

Shankar S, Hershberger CD, Chakrabarty AM
The nucleoside diphosphate kinase of Mycobacterium smegmatis: identification of proteins that modulate specificity of nucleoside triphosphate synthesis by the enzyme.
Mol Microbiol. 1997; 24: 477-87
Display abstract
We report the purification and characterization of the enzyme nucleoside diphosphate kinase (Ndk) from Mycobacterium smegmatis. The N-terminus of the enzyme was blocked but an internal sequence showed approx. 70% homology with the same enzymes from Pseudomonas aeruginosa and Escherichia coli. immobilization of the mycobacterial nucleoside diphosphate kinase on a Sepharose 4 B matrix and passing the total cell extract through it revealed four proteins (P70, P65, P60, and P50, respectively) of M(r) 70 kDa, 65 kDa, 60 kDa and 50 kDa that were retained by the column. While the proteins of M(r) 70 kDa and 50 kDa modulated the activity of Ndk directing it towards GTP synthesis, the 60 kDa protein channelled the specificity of Ndk entirely towards CTP synthesis. The 65 kDa protein modulated the specificity of Ndk directing it entirely towards UTP synthesis. The specificity for such mycobacterial proteins towards NTP synthesis is retained when they are complexed with P. aeruginosa Ndk. We further demonstrate that the P70 protein is pyruvate kinase and that each of the four proteins forms a complex with Ndk and alters its substrate specificity. Given the ubiquitous nature of Ndk in the living cell and its role in maintaining correct ratios of intracellular nucleoside triphosphates, the implications of the occurrence of these complexes have been discussed in relation to the precursor pool for cell wall biosynthesis as well as RNA/DNA synthesis.

Kimura N et al.
[Molecular mechanism of multifunctional protein, NDP kinase/nm 23]
Tanpakushitsu Kakusan Koso. 1997; 42: 745-60

Ohtsuki K, Shintani S, Kimura N, Matsumura T
Immunohistochemical study on the nm23 gene produce (NDP kinase) in oral squamous cell carcinoma.
Oral Oncol. 1997; 33: 237-9
Display abstract
The nm23 gene is a potential metastasis suppressor gene originally identified using a murine melanoma cell line. Immunohistochemical investigation on the role of nm23 gene product (nucleoside diphosphate kinase: NDPK) in squamous cell carcinomas of the oral cavity was performed and compared to clinical and histopathological factors. The stainability of NDPK did not correlate to tumour extent, differentiation and mode of invasion. However, the NDPK positive group tended to have a lower frequency of lymph node metastasis, and a better prognosis than the NDPK negative group. Consequently, nm23/NDPK is suggested to be a metastasis suppressor factor that may be useful for predicting tumour metastasis and prognosis in oral squamous cell carcinoma.

Ouatas T, Abdallah B, Gasmi L, Bourdais J, Postel E, Mazabraud A
Three different genes encode NM23/nucleoside diphosphate kinases in Xenopus laevis.
Gene. 1997; 194: 215-25
Display abstract
Nucleoside diphosphate kinases (NDPKs) catalyse the phosphorylation of nucleoside diphosphates. In mammals, the functional enzyme is a hexamer composed of different amounts of two homologous acidic (A) and basic (B) subunits encoded by separate genes. In prokaryotes and invertebrate eukaryotes, only one cytoplasmic enzyme has been isolated. Other genes encoding chloroplastic and mitochondrial forms as well as related proteins have been cloned. Here, we show that in Xenopus laevis, as in mammals, the cytoplasmic NDPK is encoded by several homologous genes. With Xenopus laevis being a pseudotetraploid species, each monomer is encoded by two genes. The amino acid sequences are very similar, and all the differences concern amino acids located at the outer surface of the hexameric enzyme. The Xenopus genes share 82-87% identity with their human counterparts. Interestingly, in vitro, the Xenopus X1 enzyme binds to a specific nuclease hypersensitive element (NHE) of the human c-myc promoter, as does its human counterpart. X1 also binds to a single-stranded (CT)(n) dinucleotide repeat. The NHE is present in the coding strand of a pyrimidine-rich region of the 3' non-coding sequence of the Xenopus NDPK genes. We propose that NDPK is indeed able to bind to its own mRNA and prevent polyadenylation at the normal position. This could provide an autoregulatory translation mechanism. A phylogenetic tree of the vertebrate NDPK sequences supports the idea that in amphibians, as in mammals, gene duplication has resulted in functional diversification.

Lambeth DO, Mehus JG, Ivey MA, Milavetz BI
Characterization and cloning of a nucleoside-diphosphate kinase targeted to matrix of mitochondria in pigeon.
J Biol Chem. 1997; 272: 24604-11
Display abstract
Nucleoside-diphosphate kinase (NDP kinase) from the matrix space of mitochondria in pigeon liver was purified to homogeneity. Degenerate oligonucleotide primers to the N-terminal sequence of the purified protein and the region containing the active site histidine were used in reverse transcriptase-polymerase chain reaction to obtain a major portion of the coding sequence for the mature protein. The sequences of the C and N termini of the mature protein, and eight residues in the signal peptide, were obtained by rapid amplification of cDNA end procedures. The entire coding sequence of a cytosolic form of NDP kinase was also determined. Both isoforms, which share 53% sequence identity, possess the characteristically conserved regions of known NDP kinases. The mature mitochondrial NDP kinase protein migrates in molecular sieving columns with an apparent molecular mass of about 66 kDa. It shows very high thermal stability even though it lacks the proline residue in the killer of prune loop, and the Tyr/Glu C termini that are important in stabilizing other NDP kinases. The affinity of the mitochondrial isoform for adenine and guanine nucleotides is much higher than for pyrimidine nucleotides, but the enzyme is especially susceptible to substrate inhibition by GDP. Semi-quantitative reverse transcriptase-polymerase chain reaction showed that the relative levels of expression of the mitochondrial isoform are liver > kidney >> heart = brain > breast muscle. The cytosolic isoform is strongly and approximately equally expressed in these same five tissues. This work is the first characterization of a NDP kinase isoform that is found in the matrix space of mitochondria.

Bodey B, Bodey B Jr, Groger AM, Siegel SE, Kaiser HE
Nm23/nucleoside diphosphate (NDP) kinase expression in human malignant melanomas: significance and implications in tumor biology.
Anticancer Res. 1997; 17: 505-11
Display abstract
The nm23/NDP kinase gene located on chromosome 17q has been proposed as metastasis suppressor gene in a variety of tumor types. Nm23 was initially isolated from the highly metastatic murine K-1735 melanoma cell line and levels of nm23 have been found to correlate inversely with metastatic potential in some tumors, but not in others. In the present immunocytochemical study, we investigated nm23 protein expression in 30 primary cutaneous malignant melanomas (CMs) and 10 metastases of malignant cutaneous melanomas (MMCMs) which had already metastasized to a distant site. We employed a sensitive, indirect, four to six step alkaline phosphatase conjugated biotin-streptavidin based immunocytochemical technique using the anti-nm23 affinity purified rabbit anti-human polyclonal antibody on formalin fixed paraffin embedded tissue sections of the malignant melanomas. We found nm23 expression in 24 out of 30 CMs with between 10% and 50% of the melanoma cells exhibiting immunoreactivity with the employed antibody. None of the ten MCMMs expressed nm23. As we described in a previous article (48), malignant melanoma is characterized by a high degree of cellular immunophenotype heterogeneity. In further support of this observation, we observed a diverse level of nm23, staining intensity in the cell subpopulations which comprises the tumor microenvironment. Nm23/NDP kinase has a diverse array of biological functions including roles in signal transduction and microtubule assembly. In our opinion, the many roles of nm23/NDP kinase are mainly involved in cell division and this may be the underlying reason that levels of this protein do not truly correlate with metastatic potential. Therefore, nm23 protein levels may correlate well with proliferative rate and degree of tumor specific dedifferentiation which are important parameters to be established in the early diagnosis, monitoring of neoplasma progression and efficacy of employed clinical trials, and the determination of prognosis of every neoplastic disease.

Xu YW, Morera S, Janin J, Cherfils J
AlF3 mimics the transition state of protein phosphorylation in the crystal structure of nucleoside diphosphate kinase and MgADP.
Proc Natl Acad Sci U S A. 1997; 94: 3579-83
Display abstract
Nucleoside diphosphate kinase reversibly transfers the gamma-phosphate of ATP onto its active site histidine. We have investigated the transition state of histidine phosphorylation with the high-resolution crystal structures of the enzyme from Dictyostelium discoideum with MgADP and either aluminium or beryllium fluoride. The bound aluminium fluoride species is the neutral species AlF3 and not the more common AlF4-. AlF3 forms a trigonal bipyramid that makes it an accurate analog of the transition state of the gamma-phosphate of ATP undergoing transfer to the catalytic histidine. Its axial ligands are a histidine nitrogen and a beta-phosphate oxygen. Beryllium fluoride also binds at the same position and with the same ligands but in a tetrahedral geometry resembling the Michaelis complex rather than the transition state. The two x-ray structures show explicit enzyme-substrate interactions that discriminate between the ground and the transition states of the reaction. They also illustrate the partially dissociative geometry of the transition state of phosphoryl transfer and demonstrate the potential applications of metallofluorides for the study of kinase mechanisms.

Heo YJ, Kim SY, Kim E, Lee KJ
Quaternary structural analysis of nucleoside diphosphate kinases using capillary electrophoresis.
J Chromatogr A. 1997; 781: 251-61
Display abstract
Capillary electrophoresis was used to monitor the quaternary structure and structural changes by denaturants of nucleoside diphosphate kinase (NDP kinase). NDP kinase from human erythrocyte consists of two kinds of polypeptide chains: A (Nm23-H1) and B (Nm23-H2), which are the products of nm23-H1 and -H2 genes, respectively. Structural characteristics of native NDP kinase, recombinant Nm23-H1 and -H2 were examined using capillary electrophoresis employing high pH running buffer in an uncoated fused-silica capillary as a separation column. The results were compared with those of the conventional sodium dodecyl sulfate and non-denaturing polyacrylamide gel electrophoresis. It shows that capillary electrophoresis is a possible tool to investigate protein structure, including the quaternary structure. This protocol was applied to the investigation of the denaturation process of each enzyme by denaturants (6 M urea or heating to 70 degrees C) and to the monitoring of the interaction between denatured Nm23-H1 and -H2. The structural information of NDP kinase obtained by analysis using capillary electrophoresis is valuable for understanding its suggested biological function as a tumor metastasis suppressor and c-myc transcription factor, etc.

Mesnildrey S, Agou F, Veron M
The in vitro DNA binding properties of NDP kinase are related to its oligomeric state.
FEBS Lett. 1997; 418: 53-7
Display abstract
Genetic and biochemical evidences suggest that the enzymatic activity of NDP kinase is necessary but not sufficient for its biological function. While the human NDPK-B binds specifically single-strand polypyrimidines sequences, the hexameric enzyme from Dictyostelium does not. We demonstrated by electrophoretic mobility shift assay and filter binding assay that a dimeric mutant from Dictyostelium binds to an oligodesoxynucleotide while the wild-type does not. These data suggest that the differences in the DNA binding properties of several eucaryotic NDP kinases might be correlated to the differences in the stability of their hexameric structure.

Cipollini G et al.
Down-regulation of the nm23.h1 gene inhibits cell proliferation.
Int J Cancer. 1997; 73: 297-302
Display abstract
nm23 gene expression is strictly related to the state of cell growth. The level of its expression parallels the fraction of thymidine-incorporating cells (S-phase cells) in neoplastic mammary tissues and in the synchronously cycling fraction of MCF 1OA cells. nm23.h1 reaches a peak of expression in the S-phase, and is present at very low level during the GO/G1 phase. Two strategies are used to demonstrate the direct involvement of the nm23.h1 gene in the process of cell proliferation. The first consists of transient inhibition of nm23.h1 expression by using anti-sense oligonucleotide treatment; weak inhibitory effect on cell proliferation is observed. The second strategy involves the stable inhibition of nm23.h1 expression by transfection of MCF1OA cells with a plasmid vector expressing the human nm23.h1 anti-sense mRNA. The anti-sense-transfected cells show consistently slower proliferative activity than the control.

Xu Y et al.
X-ray analysis of azido-thymidine diphosphate binding to nucleoside diphosphate kinase.
Proc Natl Acad Sci U S A. 1997; 94: 7162-5
Display abstract
To be effective as antiviral agent, AZT (3'-azido-3'-deoxythymidine) must be converted to a triphosphate derivative by cellular kinases. The conversion is inefficient and, to understand why AZT diphosphate is a poor substrate of nucleoside diphosphate (NDP) kinase, we determined a 2.3-A x-ray structure of a complex with the N119A point mutant of Dictyostelium NDP kinase. It shows that the analog binds at the same site and, except for the sugar ring pucker which is different, binds in the same way as the natural substrate thymidine diphosphate. However, the azido group that replaces the 3'OH of the deoxyribose in AZT displaces a lysine side chain involved in catalysis. Moreover, it is unable to make an internal hydrogen bond to the oxygen bridging the beta- and gamma-phosphate, which plays an important part in phosphate transfer.

Anciaux K, Van Dommelen K, Willems R, Roymans D, Slegers H
Inhibition of nucleoside diphosphate kinase (NDPK/nm23) by cAMP analogues.
FEBS Lett. 1997; 400: 75-9
Display abstract
Nucleoside diphosphate kinase (NDPK/nm23) ATP/GDP phosphotransferase activity and serine autophosphorylation is inhibited by N6-mbcAMP, 8-ClcAMP and 8-BrcAMP. Inhibition of the enzymatic activity largely depends on the concentration of ATP and becomes significant at ATP concentrations up to 0.5 mM and at effector concentrations measured in C6 cells stimulated with 1 mM cAMP analogue. N6-mbcAMP is a substrate of the enzyme. DbcAMP and 0'2-mbcAMP, cAMP analogues with a modified 0'2-ribose, did not affect the NDPK activity. Cyclic AMP is only a moderate inhibitor of NDPK even at low ATP concentrations. Possible inhibitory effects of cAMP and cAMP analogues on reported extra- and intracellular functions of NDPK/nm23 are discussed.

Kim SY et al.
Rapid purification and characterization of nucleoside diphosphate kinase isoforms using ATP-sepharose affinity column chromatography.
Mol Cells. 1997; 7: 630-4
Display abstract
Nucleoside diphosphate kinases (NDP kinases), products of the nm23 gene, catalyze the transfer of the terminal phosphate group of the nucleoside triphosphate to the corresponding diphosphate and may be involved in tumor metastasis suppression, development, and signal transduction. NDP kinase from various sources including human erythrocytes, rat brain tissue and E. coli strain BL21 transformed with pET3C expression plasmids containing nm23-H1 or nm23-H2, were purified in one step to homogeneity using ATP-sepharose affinity column chromatography. This method was applicable for the purification of various NDP kinases which show the same enzymatic activity and immunodetection, but have various molecular weight and quaternary structures.

Aguado-Velasco C, Veron M, Rambow JA, Kuczmarski ER
NDP kinase can modulate contraction of Dictyostelium cytoskeletons.
Cell Motil Cytoskeleton. 1996; 34: 194-205
Display abstract
Extraction of Dictyostelium amoebae with Triton X-100 produces robust cytoskeletons composed mainly of actin and myosin II. These cytoskeletons rapidly contract when mixed with Mg-ATP in simple buffers. The Triton-soluble fraction was found to contain a GTP-dependent activity that prevented contraction by Mg-ATP. This activity was purified, and identified, as nucleoside diphosphate kinase (NDP kinase). The apparent inhibition resulted from pre-contraction of the cytoskeletons. Tightly bound cytoskeletal ADP was presumably phosphorylated, and the resulting ATP powered contraction. NDP kinase appeared to be unique in this capacity, since other regenerating systems did not cause pre-contraction. Reconstitution experiments demonstrated that the kinase must be in physical contact with the cytoskeleton. These results suggest that Dictyostelium NDP kinase is able to channel ATP to the myosin molecule, and this could play a role in directly regulating cytoskeletal contraction or in facilitating contraction under conditions where intracellular ATP concentrations are low. This ability to modulate cytoskeletal contraction could help to explain observations in other systems whereby defects in NDP kinase result in abnormal development or changes in the metastatic potential of cancer cells.

Bourdais J et al.
Cellular phosphorylation of anti-HIV nucleosides. Role of nucleoside diphosphate kinase.
J Biol Chem. 1996; 271: 7887-90
Display abstract
Nucleotide analogs are widely used in antiviral therapy and particularly against AIDS. Delivered to the cell as nucleosides, they are phosphorylated into their active triphospho derivative form by cellular kinases from the host. The last step in this series of phosphorylations is performed by nucleoside diphosphate (NDP) kinase, an enzyme that can use both purine or pyrimidine and oxy- or deoxynucleotides as substrates. Using pure recombinant human NDP kinase type B (product of the gene nm23-H2), we have characterized the kinetic parameters of several nucleotide analogs for this enzyme. Contrary to what is generally assumed, diphospho- and triphospho- derivatives of azidothymidine as well as of dideoxyadenosine and dideoxythymidine are very poor substrates for NDP kinase. The rate of phosphorylation of these analogs varies between 0.05% and 0.5%, as compared to the corresponding natural nucleotide, a result that is not due to the inability of the analogs to bind to the enzyme. Using the data from the high resolution crystal structure of NDP kinase, we provide an interpretation of these results based on the crucial role played by the 3'-OH moiety of the nucleotide in catalysis.

Biondi RM et al.
Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture.
FEBS Lett. 1996; 399: 183-7
Display abstract
Although a number of nucleoside diphosphate kinases (NDPKs) have been reported to act as inhibitors of metastasis or as a transcription factor in mammals, it is not known whether these functions are linked to their enzymatic activity or how this protein is regulated. In this report, we show that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated on histidine residues, however, only the B isoform appeared to be serine phosphorylated.

Gazzeri S et al.
Overexpression of nucleoside diphosphate/kinase A/nm23-H1 protein in human lung tumors: association with tumor progression in squamous carcinoma.
Lab Invest. 1996; 74: 158-67
Display abstract
Levels of nm23-H1/nucleoside diphosphate/kinase A expression have been reported to correlate inversely with metastatic potential in some tumors but not in others. To clarify the role of nm23 in lung carcinoma, the genetic abnormalities of nucleoside diphosphate/kinase A/nm23-H1 were investigated at the DNA and protein levels. A series of 104 human lung tumors (42 neuroendocrine (NE) and 62 non-NE tumors) was analyzed for nm23-H1 protein expression by immunohistochemistry using one polyclonal and two monoclonal Ab and for genomic alterations using Southern blotting and single-strand conformation polymorphism. Overexpression of the nm23-H1 protein relative to the normal lung epithelia (pneumocyte and bronchial epithelial cells) was observed in 83% (35/42) of NE carcinomas and in 89% (55/62) of non-NE carcinomas. Eight of nine carcinoids exhibited an increased expression of nm23-H1 protein, suggesting that this overexpression of the nm23 protein is necessary for proliferation in any tumors. No significant correlation was found between nm23 staining and any clinicopathologic parameters in NE carcinoma or in adenocarcinoma. In squamous carcinoma, high levels of nm23-H1 protein expression were associated with tumor stage (p = 0.0036). Allelic deletion or genetic amplification was never found. No altered mobility was detected using single-strand conformation polymorphism analysis. These data show that nm23-H1 protein is overexpressed in a large number of lung tumors of all histologic types, in association with advanced tumor stage in squamous carcinoma. They also suggest that nm23-H1 might play a role in the progression of lung tumors rather than in antimetastatic function.

Okada K, Shiku H
[Expression of nm23/NDP kinase and cancer metastasis]
Nippon Rinsho. 1996; 54: 845-53
Display abstract
The nm23 gene was originally identified by differential hybridization between low and highly metastatic murine melanoma lines. The nm23 protein is identical with NDP kinase and there are at least two isotypes in several mammalian species such as human, mice and rats. Reduced expression of nm23/NDP kinase gene has been reported of association with highly metastatic characteristics in several experimental tumors and in certain types of human cancer. Detailed mechanisms of nm23 molecules in metastasis suppression remain unknown. The nm23/NDP kinase appears to participate to multiple cellular functions such as differentiation, proliferation and development. In this article, we reviewed recent reports on nm23/NDP kinase.

Postel EH, Weiss VH, Beneken J, Kirtane A
Mutational analysis of NM23-H2/NDP kinase identifies the structural domains critical to recognition of a c-myc regulatory element.
Proc Natl Acad Sci U S A. 1996; 93: 6892-7
Display abstract
NM23-H2, a presumed regulator of tumor metastasis in humans, is a hexameric protein with both enzymatic (NDP kinase) and regulatory (transcriptional activation) activity. While the structure and catalytic mechanisms have been well characterized, the mode of DNA binding is not known. We examined this latter function in a site-directed mutational study and identified residues and domains essential for the recognition of a c-myc regulatory sequence. Three amino acids, Arg-34, Asn-69, and Lys-135, were found among 30 possibilities to be critical for DNA binding. Two of these, Asn-69 and Lys-135, are not conserved between NM23 variants differing in DNA-binding potential, suggesting that DNA recognition resides partly in nonconserved amino acids. All three DNA-binding defective mutant proteins are active enzymatically and appear to be stable hexamers, suggesting that they perform at the level of DNA recognition and that separate functional domains exist for enzyme catalysis and DNA binding. In the context of the known crystal structure of NM23-H2, the DNA-binding residues are located within distinct structural motifs in the monomer, which are exposed to the surface near the 2-fold axis of adjacent subunits in the hexamer. These findings are explained by a model in which NM23-H2 binds DNA with a combinatorial surface consisting of the "outer" face of the dimer. Chemical crosslinking data support a dimeric DNA-binding mode by NM23-H2.

Schaertl S
Quaternary structure of human nucleoside diphosphate kinase isoforms HA and HB in solution.
FEBS Lett. 1996; 394: 316-20
Display abstract
Human isoforms of nucleoside diphosphate kinase, NDPK-HA and NDPK-HB, have been expressed in E. coli and purified. Their apparent molecular masses have been determined by FPLC gel filtration. Absolute molecular masses were measured by equilibrium ultracentrifugation and sedimentation coefficients determined from the sedimentation velocity. Under near-physiological conditions, NDPK-HA has a mass of 101 +/- 3 kDa, close to that calculated for a hexamer (102.11 kDa), whilst NDPK-HB has a mass of 71 +/- 3 kDa, close to a tetramer (68.67 kDa). The sedimentation coefficients, 5.15 +/- 0.2 and 3.41 +/- 0.1 x 10(-13) s, for HA and HB also indicate a hexamer and a tetramer respectively. This suggests, although the crystal structure shows a hexameric quaternary arrangement [Webb et al. (1995) J. Mol. Biol. 251, 574-587], that NDPK-HB forms tetramers in solution like bacterial NDPK [Williams et al. (1993) J. Mol. Biol. 234, 1230-1247].

Ann KS, Nelson DL
A nucleoside diphosphate kinase from Paramecium tetraurelia with protein kinase activity.
J Eukaryot Microbiol. 1996; 43: 365-72
Display abstract
Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound alpha-labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid-stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross-reacted with 16- to 20-kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase-like awd protein of Drosophila and Nm23 protein of humans.

Giartosio A et al.
Thermal stability of hexameric and tetrameric nucleoside diphosphate kinases. Effect of subunit interaction.
J Biol Chem. 1996; 271: 17845-51
Display abstract
The eukaryotic nucleoside diphosphate (NDP) kinases are hexamers, while the bacterial NDP kinases are tetramers made of small, single domain subunits. These enzymes represent an ideal model for studying the effect of subunit interaction on protein stability. The thermostability of NDP kinases of each class was studied by differential scanning calorimetry and biochemical methods. The hexameric NDP kinase from Dictyostelium discoideum displays one single, irreversible differential scanning calorimetry peak (Tm 62 degrees C) over a broad protein concentration, indicating a single step denaturation. The thermal stability of the protein was increased by ADP. The P105G substitution, which affects a loop implicated in subunit contacts, yields a protein that reversibly dissociates to folded monomers at 38 degrees C before the irreversible denaturation occurs (Tm 47 degrees C). ADP delays the dissociation, but does not change the Tm. These data indicate a "coupling" of the quaternary structure with the tertiary structure in the wild-type, but not in the mutated protein. We describe the x-ray structure of the P105G mutant at 2.2-A resolution. It is very similar to that of the wild-type protein. Therefore, a minimal change in the structure leads to a dramatic change of protein thermostability. The NDP kinase from Escherichia coli behaves like the P105G mutant of the Dictyostelium NDP kinase. The detailed study of their thermostability is important, since biological effects of thermolabile NDP kinases have been described in several organisms.

Lu Q, Park H, Egger LA, Inouye M
Nucleoside-diphosphate kinase-mediated signal transduction via histidyl-aspartyl phosphorelay systems in Escherichia coli.
J Biol Chem. 1996; 271: 32886-93
Display abstract
Nucleoside-diphosphate kinase (NDP kinase), a key enzyme in nucleotide metabolism, is also known to be involved in growth and developmental control and tumor metastasis suppression. Interestingly, we find that coexpression of NDP kinase with Taz1, a Tar/EnvZ chimera, in the absence of its native signal, can activate a porin gene ompC-lacZ expression in Escherichia coli. Further studies show that NDP kinase can act as a protein kinase to phosphorylate histidine protein kinases such as EnvZ and CheA which are members of the His-Asp phosphorelay signal transduction systems in E. coli. Instead of ATP, the exclusive phosphodonor for histidine kinases, GTP can be utilized in vitro in the presence of NDP kinase to phosphorylate EnvZ and CheA, which then transfer the phosphoryl group to OmpR and CheY, the respective response regulators. The direct involvement of GTP for the phosphorylation of EnvZ through NDP kinase was further demonstrated by the use of a mutant EnvZ, which lost ability to be autophosphorylated with ATP. Phospho-OmpR thus formed can bind specifically to an ompF promoter sequence. These results suggest that NDP kinase may play a physiological role in signal transduction.

Deville-Bonne D, Sellam O, Merola F, Lascu I, Desmadril M, Veron M
Phosphorylation of nucleoside diphosphate kinase at the active site studied by steady-state and time-resolved fluorescence.
Biochemistry. 1996; 35: 14643-50
Display abstract
Nucleoside diphosphate (NDP) kinase is the enzyme responsible in the cell for the phosphorylation of nucleoside or deoxynucleoside diphosphates into the corresponding triphosphates at the expense of ATP. Transfer of the gamma-phosphate is very fast (turnover number above 1000 s-1) and involves the phosphorylation of a histidine residue at the active site of the enzyme. We have used intrinsic protein fluorescence of the single tryptophan of Dictyostelium discoideum NDP kinase as a sensitive probe for monitoring the interaction of the enzyme with its substrates. We demonstrate that the 20% quenching of steady-state fluorescence observed upon addition of ATP is due to formation of the phosphorylated intermediate. Time-resolved fluorescence indicates that the Trp-137 side chain is rigidly bound to the protein core with a unique lifetime of 4.5 ns for the free enzyme at 20 degrees C and that it remains tightly immobilized during the time course of the reaction. Phosphorylation of this catalytic residue (His-122) in the presence of ATP induces a similar decrease in mean lifetime, due to the splitting of the signal and the appearance of a shorter decay. This splitting is discussed in terms of a slow conformational equilibrium. We demonstrate that, in the wild-type enzyme, the conserved His-55 quenches the fluorescence of Trp-137 as the H55A mutant protein fluorescence displays an increase in quantum yield. Even though H55A mutant enzyme is active, the absence of the imidazole ring prevents the detection of the phosphorylated state of His-122 by Trp-137. We conclude that His-55 serves as a relay between His-122 and Trp-137.

Yang LM, Lamppa GK
Rapid purification of a chloroplast nucleoside diphosphate kinase using CoA-affinity chromatography.
Biochim Biophys Acta. 1996; 1294: 99-102
Display abstract
An 18 kDa protein from spinach chloroplasts was purified in one step to homogeneity using CoA-affinity chromatography. Its N-terminal sequence was identical to spinach nucleoside diphosphate kinase II (NDPK II). The kinase was isolated as a approximately 100 kDa complex. Immunoblotting detected NDPKII in plastids from leaves, roots, seeds and male flowers. NDPK I, an isoform of NDPK II, was not found in chloroplast soluble extracts, whereas NDPK III was present.

Chang CL, Strahler JR, Thoraval DH, Qian MG, Hinderer R, Hanash SM
A nucleoside diphosphate kinase A (nm23-H1) serine 120-->glycine substitution in advanced stage neuroblastoma affects enzyme stability and alters protein-protein interaction.
Oncogene. 1996; 12: 659-67
Display abstract
A high level of nucleoside diphosphate kinase A (NDPK A/nm23-H1) in neuroblastoma is associated with advanced stage disease. We have also found a serine 120-->glycine substitution in NDPK A and/or amplification of the nm23-H1 gene in advanced stage neuroblastomas. Serine 120, a highly conserved residue, is located in proximity to histidine 118 which forms a phosphorylated intermediate essential for NDPK activity. The effect of Ser120-->Gly substitution on the biochemical properties of NDPK A was investigated. Phosphate-transferase activity was lower in the recombinant mutant NDPK A and in the immunoprecipitated complex consisting of NDPK A and NDPK B prepared from a neuroblastoma tumor containing the mutation, relative to the wild-type. There was a significant decrease in the enzyme stability toward urea- or temperature-induced denaturation for the recombinant mutant NDPK A and in an immunoprecipitate from a tumor containing the mutation. Recombinant NDPK A containing the Ser120-->Gly mutation exhibited reduced hexameric and increased dimeric oligomerization relative to the wild-type. Moreover a 28 kDa cellular protein was detected, that co-precipitated with the mutant but not wild-type NDPK A. The altered properties of the mutant protein may have relevance to a role for NDPK A in neuroblastoma progression.

Hamada T, Tanaka N, Noguchi T, Kimura N, Hasunuma K
Phytochrome regulates phosphorylation of a protein with characteristics of a nucleoside diphosphate kinase in the crude membrane fraction from stem sections of etiolated pea seedlings.
J Photochem Photobiol B. 1996; 33: 143-51
Display abstract
The molecular mechanism of light signal perception via phytochrome was analysed using the third internodes of etiolated pea seedlings irradiated with red or red followed by far-red light. A crude membrane fraction prepared from the tissue was labelled by [gamma-32P]ATP at 4 x 10(-8) M for 15 s at 0 degree C, and the proteins were separated by two-dimensional gel electrophoresis. The phosphorylation of a protein with a molecular mass of about 15 kDa in the crude membrane fraction increased with an increase in the intensity of red light irradiation (10, 50 and 100 mumol m-2 s-1) for 20 s. Successive irradiation with red light (100 mumol m-2 s-1 for 20 s) and subsequent irradiation with far-red light reduced the phosphorylation of the protein, depending on the intensity of the far-red light (from 0.1 to 8 mumol m-2 s-1 for 180 s). A plasma membrane purified from the crude membrane fraction from red light irradiated tissue showed a rapid phosphorylation of the 15 kDa protein by 4 x 10(-8) M [gamma-32P]ATP at 0 degree C for 7 s, and subsequent addition of ATP, GTP, ADP or GDP at 10(-5) or 10(-6) M efficiently removed the phosphoryl group of the 15 kDa protein. The 15 kDa protein was autophosphorylated in the gel following separation by sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis. The partially purified 15 kDa protein included nucleoside diphosphate kinase (NDP kinase) activity, as well as cross-reactivity with an antibody specific to rat NDP kinase as assayed by immunostaining and crossreactivity with an antibody specific to ricet NDP kinase as assayed by immunoprecipitation.

Karlsson A, Mesnildrey S, Xu Y, Morera S, Janin J, Veron M
Nucleoside diphosphate kinase. Investigation of the intersubunit contacts by site-directed mutagenesis and crystallography.
J Biol Chem. 1996; 271: 19928-34
Display abstract
NDP kinase from Dictyostelium was mutated by site-directed mutagenesis at positions indicated by structural data to be involved in the trimer interface. The mutants were substitutions at residue Pro-100 (P100S and P100G) and deletions of 1-5 residues at the C terminus. Single mutants yielded proteins that kept both activity and hexameric structure. However, they were severely affected in their stability toward temperature and urea denaturation. When the P100S mutation was combined with any of the C-terminal deletions, the enzyme lost most of its activity and dissociated into dimers. Crystallographic analysis of the P100S protein was performed at 2.6 A resolution. The x-ray structure showed no direct alteration of intersubunits contacts at residue 100, but an induced disruption of the interaction between Asp-115 and the C terminus of another subunit. The substitution of proline 100 to serine corresponds to the Killer-of-prune mutation in Drosophila. Consequences of the mutation are discussed in view of the structural and biochemical properties observed in the mutant Dictyostelium protein.

Ananvoranich S, Grandmaison J, Gulick PJ
Molecular and biochemical characterization of two nucleoside diphosphate kinase cDNA clones from Flaveria bidentis.
Genome. 1996; 39: 404-9
Display abstract
Two nucleoside diphosphate kinase cDNA clones have been isolated from Flaveria bidentis by immunoscreening of an expression library with a polyclonal antibody raised against Flaveria chloraefolia flavonol 3-sulfotransferase (F3-ST). The clones represent members of a small multigene family in this species. The nucleotide sequences of the two cDNA clones show a high degree of sequence similarity to other reported nucleoside diphosphate kinases (NDPKs), including the putative human tumor suppressor gene NM23 and the Drosophila regulatory gene. When these cDNA clones were expressed in Escherichia coli, their gene products exhibited NDPK enzymatic activity. The immunocross reaction of the clones with the antibody raised against the F3-ST suggests a common immuno-epitope and a similarity of a nucleotide binding site for the two proteins.

Inoue H, Takahashi M, Oomori A, Sekiguchi M, Yoshioka T
A novel function for nucleoside diphosphate kinase in Drosophila.
Biochem Biophys Res Commun. 1996; 218: 887-92
Display abstract
Nucleoside diphosphate (NDP) kinase is an enzyme that transfers the gamma-phosphate of nucleoside triphosphates to nucleoside diphosphates. Besides this well-defined role, recent evidence suggests that NDP kinase may be implicated in a wide variety of essential cellular processes. In this paper, we showed that the NDP kinase of Drosophila exhibited protein kinase activity as well as autophosphorylation. Ovalbumin was phosphorylated with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) or ATP. Protein kinase activity was not detected in NDP kinase mutant, abnormal wing discs (awd). These results suggest that this activity could be one of the functions of NDP kinase essential for normal fly development, since awd gene is lethal.

Biondi RM, Walz K, Issinger OG, Engel M, Passeron S
Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase.
Anal Biochem. 1996; 242: 165-71
Display abstract
We have critically analyzed current methodologies for distinguishing histidine and serine phosphorylated residues in proteins and report a simple technique that assures a reliable discrimination. Electro-transfer of a phosphorylated enzyme to Immobilon membranes and its treatment at pH 1 and 14 in buffers containing 5% methanol allows unambiguous distinction between serine/threonine and histidine phosphorylation (O-phosphomonoesters and phosphoramide, respectively) since under these conditions only one type of residue is dephosphorylated. The addition of 5% methanol to all buffers was indispensable to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic phosphate transfer activity of nucleoside diphosphate kinase (NDP kinase). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesis has been advanced that this nonhistidine phosphorylation may play an important role in NDP kinase cellular function, signaling the suppression of metastasis in the case of human NDP kinase A. Using this improved method, we show that human, Escherichia coli and Candida albicans NDP kinases are only autophosphorylated on histidine residues. In addition, we present evidence that the presence of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe that the technique will be generally useful in histidine phosphorylation screenings.

Fukuda M et al.
Decreased expression of nucleoside diphosphate kinase alpha isoform, an nm23-H2 gene homolog, is associated with metastatic potential of rat mammary-adenocarcinoma cells.
Int J Cancer. 1996; 65: 531-7
Display abstract
The nm23 gene [encoding nucleoside diphosphate kinase (NDPK)] may act as a metastasis suppressor in certain tumor cells. We investigated the role of NDPK isoforms (alpha and beta) in the metastatic processes, using rat mammary-adenocarcinoma cell lines of poor (MTC) and high (MTLn3) spontaneous metastatic potential respectively. In these cell lines, as in most rat tissues, the alpha isoform (nm23-H2 homolog) was more highly expressed than the beta isoform (nm23-H1 homolog) at the mRNA and protein levels. When examined by Northern- and Western-blot analyses, expression of the 2 isoforms was reduced in highly metastatic MTLn3 cells compared with poorly metastatic MTC cells. The reduced expression was also associated with diminished NDPK-enzyme activity in the cell extracts. Southern-blot and RT-PCR-SSCP analyses suggested that the 2 genes were not grossly altered or mutated in their translation regions. MTLn3 cell clones transfected with NDPKalpha or NDPKbeta cDNA were all tumorigenic when implanted into the mammary fat pad of syngeneic rats. Among those, only clones transfected with the NDPKalpha gene exhibited reduced lung metastasis in a spontaneous metastasis assay.

Konno R, Sato S, Ito K, Yajima A
[Immunohistochemical staining of nm23/nucleotide diphosphate kinase in invasive cervical carcinoma tissue and metastatic lymph nodes]
Nippon Sanka Fujinka Gakkai Zasshi. 1996; 48: 279-80

Brodbeck M, Rohling A, Wohlleben W, Thompson CJ, Susstrunk U
Nucleoside-diphosphate kinase from Streptomyces coelicolor.
Eur J Biochem. 1996; 239: 208-13
Display abstract
Nucleoside-diphosphate (NDP) kinase was purified from crude extracts of Streptomyces coelicolor to over 90% homogeneity in a single step using an adenosine 3',5'-cyclic monophosphate (cAMP) binding column. The specific activity of protein in the fraction eluted with cAMP (400 U/mg) was about 3600-fold higher than that in the crude extract. This enzyme was autophosphorylated in the presence of [y-32P]ATP. The high-energy phosphoenzyme intermediate was stable in alkali and highly labile in acid; this suggests the presence of an N-phosphate amino acid (most probably a histidine residue). A tetrameric form of the 15-kDa protein was suggested by its apparent molecular mass (66 kDa) on a gel filtration column. The measured Michaelis constant (Km) for ATP was 85 microM. The IC50 for cAMP of 6 mM suggested weak competitive inhibition. However, no evidence that cAMP acts as an allosteric effector was obtained. The ndk gene from S. coelicolor was isolated and sequenced. The deduced amino acid sequence was very similar to other NDP kinases. However, unique characteristics were also noted, including a truncated C-terminus that makes it one of the smallest NDP kinases reported in the literature.

Lu Q, Inouye M
Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.
Proc Natl Acad Sci U S A. 1996; 93: 5720-5
Display abstract
Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase.

Sonnemann J, Mutzel R
Cytosolic nucleoside diphosphate kinase associated with the translation apparatus may provide GTP for protein synthesis.
Biochem Biophys Res Commun. 1995; 209: 490-6
Display abstract
Elongation of nascent polypeptides in a Dictyostelium discoideum in vitro translation system did not require the addition of ATP and GTP when creatine phosphate and creatine phosphokinase were present. However, depletion of the exogenous energy supply completely abolished incorporation of amino acids. Addition of dTTP, a nucleoside triphosphate that can be utilized by nucleoside diphosphate kinase (NDP kinase) to phosphorylate endogenous ADP and GDP, partially restored protein synthesis. Dictyostelium ribosomes were found to contain NDP kinase activity that could not be released by 1 M KCl. Thermal denaturation studies, specific inhibition with antibodies, and Western blotting identify the activity as cytosolic NDP kinase. These data support the idea that GTP can be fed into the translation machinery efficiently by NDP kinase associated with active ribosomes.

Almaula N, Lu Q, Delgado J, Belkin S, Inouye M
Nucleoside diphosphate kinase from Escherichia coli.
J Bacteriol. 1995; 177: 2524-9
Display abstract
Nucleoside diphosphate (NDP) kinase from Escherichia coli was purified to homogeneity and was crystallized. Gel filtration analysis of the purified enzyme indicated that it forms a tetramer. The enzyme was phosphorylated with [gamma-32P]ATP, and the pH stability profile of the phosphoenzyme indicated that two different amino acid residues were phosphorylated. Both a histidine residue and serine residues, including Ser-119 and Ser-121, appear to be phosphorylated. A Ser119Ala/Ser121Ala double mutant (i.e., with a Ser-to-Ala double mutation at positions 119 and 121), as well as Ser119Ala and Ser121Ala mutants, was isolated. All of these retained NDP kinase activity; also, both the Ser119Ala and Ser121Ala mutants could still be autophosphorylated. In the case of the double mutant, a slight autophosphorylation activity, which was resistant to acid treatment, was still detected, indicating that an additional minor autophosphorylation site besides His-117 exists. These results are discussed in light of the recent report of N. J. MacDonald et al. on the autophosphorylation of human NDP kinase (J. Biol. Chem. 268:25780-25789, 1993).

Morera S, Chiadmi M, LeBras G, Lascu I, Janin J
Mechanism of phosphate transfer by nucleoside diphosphate kinase: X-ray structures of the phosphohistidine intermediate of the enzymes from Drosophila and Dictyostelium.
Biochemistry. 1995; 34: 11062-70
Display abstract
Nucleoside diphosphate kinase (NDP kinase) has a ping-pong mechanism with a phosphohistidine intermediate. Crystals of the enzymes from Dictyostelium discoideum and from Drosophila melanogaster were treated with phosphoramidate, and their X-ray structures were determined at 2.1 and 2.2 A resolution, respectively. The atomic models, refined to R factors below 20%, show no conformation change relative to the free proteins. In both enzymes, the active site histidine was phosphorylated on N delta, and it was the only site of phosphorylation. The phosphate group interacts with the hydroxyl group of Tyr56 and with protein-bound water molecules. Its environment is compared with that of phosphohistidines in succinyl-CoA synthetase and in phosphocarrier proteins. The X-ray structures of phosphorylated NDP kinase and of previously determined complexes with nucleoside diphosphates provide a basis for modeling the Michaelis complex with a nucleoside triphosphate, that of the phosphorylated protein with a nucleoside diphosphate, and the transition state of the phosphate transfer reaction in which the gamma-phosphate is pentacoordinated.

Engel M et al.
A novel serine/threonine-specific protein phosphotransferase activity of Nm23/nucleoside-diphosphate kinase.
Eur J Biochem. 1995; 234: 200-7
Display abstract
Two human nm23 genes have been identified, designated nm23-H1 and nm23-H2, which encode the 88% identical nucleoside-diphosphate kinase (NDPK) A and NDPK B polypeptides, respectively. The nm23-H1 gene product has been shown to play a functional role in the suppression of tumor metastasis. The Nm23 proteins/NDPK are highly conserved throughout evolution and are implicated in controlling cellular differentiation and development in various species, while the underlying mechanisms remain undefined. Neither the NDPK activity nor the DNA-binding activity, identified recently for NDPK B, can satisfactory explain the regulatory functions of Nm23. The present study provides evidence that purified Nm23 proteins are capable of transferring a phosphate group to other proteins when non-denaturing amounts of urea are present. This novel Nm23/NDPK activity was found to be specific for serine and threonine residues, and the transphosphorylation of substrate proteins occurred stoichiometrically. Because of the absence of a substrate turn-over, the novel function was termed protein phosphotransferase activity instead of protein kinase activity. It is demonstrated that urea stimulates the interaction of NDPK with other proteins. Identical phosphoprotein patterns were obtained using purified NDPK preparations from human, Drosophila, yeast and Dictyostelium in the presence of urea. Partially purified NDPK from human erythrocytes produced a similar phosphorylation pattern independent of urea addition and also acted stoichiometrically. In this preparation, a protein phosphotransferase activity of Nm23 species may possibly be generated and/or stabilized by the interaction with copurified proteins. Using different mutants of Dictyostelium NDPK it was shown that the protein phosphotransferase activity depends on the same active site as the NDPK activity. A phosphotransfer mechanism analogous to that of protein-histidine kinases is proposed, involving a high-energy phosphohistidine intermediate. Furthermore, the novel Nm23 function is compared with an apparently similar protein phosphotransferase activity which was observed previously with partially purified NDPK from different plant species.

Zhang J, Fukui T, Ichikawa A
A third type of nucleoside diphosphate kinase from spinach leaves: purification, characterization and amino-acid sequence.
Biochim Biophys Acta. 1995; 1248: 19-26
Display abstract
A third type of nucleoside diphosphate kinase (NDP kinase III), distinct from the previously described NDP kinases I and II (Nomura et al. (1991) Biochim. Biophys. Acta 1077, 47-55), was purified from spinach leaves to electrophoretic homogeneity. NDP kinase III was judged by SDS-PAGE and by gel filtration to have molecular masses of 17 kDa and 102 kDa, respectively, suggesting that it is composed of six subunits similarly to the other spinach isoforms, NDP kinases I and II. Amino-acid sequence analysis revealed the primary structure of NDP kinase III to be comprised of 153 amino-acid residues, the sequence of which exhibited 61% and 53% homology with those of NDP kinases I and II, respectively. In the reaction catalyzed by the three isoforms, the order of Km as phosphate acceptor was determined as GDP << ADP for NDP kinase III, different from those observed for NDP kinase I (ADP << GDP) and for NDP kinase II (GDP = ADP). These results suggest that the three isoforms may have distinct roles in regulating intracellular 5'-di- and 5'-triphosphonucleotide levels in spinach leaves.

Lubeck J, Soll J
Nucleoside diphosphate kinase from pea chloroplasts: purification, cDNA cloning and import into chloroplasts.
Planta. 1995; 196: 668-73
Display abstract
Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) was enriched 1900-fold from purified pea (Pisum sativum L. cv. Golf.) chloroplasts. The active enzyme preparation contained two polypeptides of apparent molecular weight 18.5 kDa and 17.4 kDa. Both proteins were enzymatically active and were recognized by an antiserum raised against NDPK from spinach chloroplasts, suggesting the existence of two isoforms in pea chloroplasts. The N-terminal protein sequence data were obtained for both polypeptides and compared with the nucleotide sequence of a cDNA clone isolated from a pea cDNA library. The analysis revealed that the two NDPK forms are encoded for by one mRNA, indicating that the lower-molecular-weight form could represent a proteolytic breakdown product of the 18.5-kDa NDPK. The pea chloroplastic NDPK is made as a larger precursor protein which is imported into chloroplasts. The NDPK precursor is then processed by the stromal processing peptidase to yield the 18.5-kDa form.

Strelkov SV, Perisic O, Webb PA, Williams RL
The 1.9 A crystal structure of a nucleoside diphosphate kinase complex with adenosine 3',5'-cyclic monophosphate: evidence for competitive inhibition.
J Mol Biol. 1995; 249: 665-74
Display abstract
The X-ray structure of Myxococcus xanthus nucleoside diphosphate (NDP) kinase complexed with adenosine 3',5'-cyclic monophosphate (cAMP) has been determined. The structure was solved by difference Fourier analysis. The refined structure has a crystallographic R-factor of 0.17 at 1.9 A resolution. The phosphoryl group and ribose moiety make extensive polar interactions with the protein, whereas the base interacts only with two hydrophobic residues. The comparison with the structure of the enzyme complex with the substrate adenosine diphosphate (ADP) reported earlier shows that cAMP and ADP interact similarly with the enzyme. The base of the cAMP is present in two conformations, syn and anti, with respect to the sugar. The syn conformer is dominant. Based on the effect of cAMP on phosphorylation of the human NDP kinase NM23, it had been proposed that cAMP might interact with NDP kinase in a manner distinct from other nucleotides. However, the structure of the M. xanthus NDP kinase/cAMP complex indicates that the nucleotide is a competitive inhibitor of the enzyme and occupies the usual nucleotide site. Kinetic assays of the NDP kinase activity in the presence of cAMP were done. Their results are consistent with a competitive character of the cAMP inhibition.

Giarnieri E et al.
Overexpression of NDP kinase nm23 associated with ploidy image analysis in colorectal cancer.
Anticancer Res. 1995; 15: 2049-53
Display abstract
The nm23 gene was originally identified by differential hybridization between two murine melanoma cell sublines with low and high metastatic potential. Nm23 is localized on chromosome 17q21.3-22. Allelic deletions of chromosome 17 have been related to the progression of colorectal carcinomas. We have evaluated and compared the expression of nm23 NPD kinase protein using an immunohistochemical method and DNA ploidy evaluation with image analysis. This study was performed on 20 patients, who underwent surgery for colorectal carcinoma. Patients were followed up during the period from 1992 to 1994. Results have shown an association between the parameters obtained for the nm23 NPD kinase protein expression, and aneuploid DNA and neoplastic progression. The expression of nucleoside diphosphate (NDP) kinase mm23 has been reported to be inversely related to the metastatic potential of experimental cells in human breast cancer. A relationship between the positivity in protein expression of gene product in the allele nm23 H1 and the state of the lymph nodes has also been found.

Lu Q, Zhang X, Almaula N, Mathews CK, Inouye M
The gene for nucleoside diphosphate kinase functions as a mutator gene in Escherichia coli.
J Mol Biol. 1995; 254: 337-41
Display abstract
Nucleoside diphosphate (NDP) kinase is a key enzyme in the control of cellular concentrations of nucleoside triphosphates, and has been shown to play important roles in various cellular activities such as developmental control, signal transduction and metastasis in eukaryotic systems. In this study, the gene for NDP kinase of Escherichia coli (ndk) was disrupted and surprisingly found to be dispensable without any discernible effects on cell growth or morphology. However, a mutator phenotype was found in ndk-disruption strains; frequencies of spontaneous mutations to rifampicin resistance and nalidixic acid resistant significantly increased. A higher frequency in reversion mutations was observed with use of an amber mutation in the kanamycin-resistance gene in an ndk-disruption strain. Imbalance in dNTP pools, in particular a significant increase of the dCTP content was observed, which is likely to result in the higher spontaneous mutation rates. These results suggest that NDP kinase, although not essential, plays an important role in the appropriate balance of intracellular dNTP pools to maintain a high DNA replication fidelity. Strains with ndk- pykA- pykF- as well as ndk- scs- were constructed without any discernible effect on cell growth, indicating that there is yet another enzyme(s) catalyzing nucleoside triphosphate synthesis, in addition to NDP kinase, pyruvate kinases and succinyl CoA synthetase.

Baba H et al.
Two isotypes of murine nm23/nucleoside diphosphate kinase, nm23-M1 and nm23-M2, are involved in metastatic suppression of a murine melanoma line.
Cancer Res. 1995; 55: 1977-81
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A series of sublines of a murine melanoma B16 of C57BL/6 origin were established and examined regarding their metastatic capacity and expression of nm23. The number of pulmonary metastases developed by these sublines was inversely correlated with the expression of two isotypes of nm23, nm23-M1 and nm23-M2. The cDNAs of nm23-M1, nm23-M2, and a combination of both were transfected into the highly metastatic melanoma subline FE7, with low nm23 expression. FE7 transfectants of any of these cDNAs expressed transfected genes, and their metastatic capacity was suppressed when compared with parental FE7 or FE7 transfected with a control neo gene. These cell lines, however, did not change in terms of in vitro growth in the presence of 3 or 10% fetal bovine serum and in vivo growth when injected s.c. into C57BL/6-nu/nu mice. Similar experiments were also performed using FE7 transfectants of human nm23 genes. Transfectants of nm23-H1, nm23-H2, and their combination did not present altered metastatic potential. These findings indicated that two murine isotypes of nm23 but not those of humans are intimately related with the suppression of metastasis in the murine body.

Ghosh I, Raghavan N, FitzGerald PC, Scott AL
Nucleoside diphosphate kinase from the parasitic nematode Brugia malayi.
Gene. 1995; 164: 261-6
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Using a reverse transcription-polymerase chain reaction (RT-PCR) procedure that exploited the presence of a conserved 22-nucleotide spliced leader (SL) sequence that is trans-spliced to the 5' end of nematode transcripts, a novel Brugia malayi (Bm) infective-stage SL cDNA expression library was constructed and characterized. The library was immunoscreened with rabbit anti-infective-stage antibodies (Ab) and an immunodominant clone, BmG4-7, was identified and characterized. BmG4-7 contained a full-length cDNA that had significant sequence similarity to nucleoside diphosphate kinase (NDK)-encoding sequences reported from a number of species, including Drosophila melanogaster and humans. BmNDK was found to be constitutively transcribed during all stages of parasite development. An anti-BmNDK Ab was used to immunostain a Western blot of extracts from adult and larval parasites. The Ab specifically recognized a 17.5-kDa molecule in all of the parasite extracts. Molecular modeling of the BmNDK showed several regions surrounding the conserved catalytic site that may be important in the design of drugs specific for the disruption of NTP synthesis in filarial parasites.

Lecroisey A, Lascu I, Bominaar A, Veron M, Delepierre M
Phosphorylation mechanism of nucleoside diphosphate kinase: 31P-nuclear magnetic resonance studies.
Biochemistry. 1995; 34: 12445-50
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The phosphorylation mechanism of Dictyostelium discoideum nucleoside diphosphate (NDP) kinase was investigated by NMR. 31P chemical shifts were measured on both native and denatured enzyme. In the enzymatically phosphorylated enzyme denatured by 9 M urea or 7 M guanidine hydrochloride, the NDP kinase phosphohistidine signal appeared between the signals of N delta and N epsilon free monophosphohistidines used as reference compounds and added to the sample. A signal with the same intermediate position was also observed in the pronase digest of the alkaline-denatured phosphorylated enzyme. However, when phosphohistidines of the phosphorylated synthetic peptide pGlu-His-Gly were taken as references, the NDP kinase and the N delta peptide phosphohistidine signals were shown to be identical, providing evidence that phosphorylation occurs on the N delta of the active site histidine residue. Moreover, the rate of hydrolysis of the histidine-bound phosphate is in agreement with a modification at the N delta position. Phosphorylation of the NDP kinase by phosphoramidate provided a result similar to that of the enzymatic phosphorylation. In both cases, phosphorylation could not be detected on any amino acid other than histidine. Particularly, no phosphoserine residue was observed.

Biondi RM, Veron M, Walz K, Passeron S
Candida albicans nucleoside-diphosphate kinase: purification and characterization.
Arch Biochem Biophys. 1995; 323: 187-94
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Soluble nucleoside-diphosphate kinase (NDP kinase) from Candida albicans was purified to electrophoretic homogeneity and a partial sequence was determined. The enzyme was kinetically and physically characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single polypeptide of 17 kDa upon staining, by immunodetection with heterologous and homologous antibodies, and by autoradiography of the phosphorylated enzyme. Furthermore, isoelectric focusing of the purified native enzyme showed a single acidic band (pI 4.5). These results together with a native molecular mass of 98 kDa suggest a hexameric native enzyme composed of identical subunits. Like NDP kinases from other sources, the catalysis involves a phosphoenzyme intermediate that is rapidly formed upon incubation of the enzyme with ATP. The transfer of phosphate from phosphoprotein intermediate to nucleoside diphosphates is equally fast. Kinetic experiments indicated that GTP and ATP had the lowest Km compared to UTP, dTTP, and CTP. GDP acted as a preferred acceptor as assessed by kinetic measurements as well as by competition experiments. Experimental data are presented indicating the existence of a membrane-associated NDP kinase. Preliminary characterization of this enzyme suggests that cytosolic and membrane-associated NDP kinases are similar proteins.

Sellam O, Veron M, Hildebrandt M
Overexpression of wild-type and mutant NDP kinase in Dictyostelium discoideum.
Mol Microbiol. 1995; 16: 79-85
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Nucleoside diphosphate (NDP) kinase has a central role in the synthesis of (deoxy-)trinucleotides. In addition, mutations in the gene encoding NDP kinase have been shown to have important consequences for Drosophila development and mammalian tumorogenesis. We have overexpressed, in Dictyostelium discoideum, a genomic clone encoding the enzyme NDP kinase. The concomitant increase in the levels of RNA and enzyme activity identifies a 5' non-coding genomic region of 0.9 kb as being the complete promoter region. Overexpression of wild-type NDP kinase has no effect on development. This is also true for an inactive mutant H122C that does not have a dominant inhibitor effect. Overexpression of the P105G mutant NDP kinase, which is known to be affected in its stability in vitro, only leads to a small increase in total NDP-kinase activity. Thermal and chemical denaturation experiments demonstrate the formation of hexameric hybrids between wild-type and mutant monomers.

Zhang SQ, Hu Y, Jong AY
Temporal and spatial distributions of yeast nucleoside diphosphate kinase activities and its association with the Cdc8p.
Cell Mol Biol Res. 1995; 41: 333-46
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Nucleoside diphosphate kinase (E.C. 2.7.4.6.) is a broad substrate-specific enzyme that catalyzes the phosphorylation of nucleoside diphosphates to the corresponding triphosphates in nucleic acid biosynthesis. In this report, we investigate its spatial and temporal distributions in yeast to understand how the enzyme exerts its gene function(s). Our results show that the enzyme is predominantly cytoplasmic. A substantial amount of enzyme activity (40-50%) may be associated with the cell membrane. Less than 1% of total activity was detected in the nuclear fraction. Approximately 3% was found in the mitochondrial fraction. When yeast cultures were synchronized, we found that Saccharomyces cerevisiae nucleoside diphosphate kinase did not show cell cycle periodicity, as Schizosaccharomyces pombe enzyme did. To explore its link with DNA synthesis, we investigated its relationship with the Cdc8p (dTMP kinase). We demonstrated a physical interaction between these proteins in vitro, as evidenced that the GST:Cdc8p protein affinity column could retain a subpopulation of nucleoside diphosphate kinase activity from yeast crude extract. Furthermore, when GST:Cdc8p protein was expressed in yeast, the protein could bind to the glutathione-agarose, along with nucleoside diphosphate kinase, suggesting that there is an interaction between GST:Cdc8p and nucleoside diphosphate kinase in vivo. Our results provide evidence for at least a two-enzyme complex that may well facilitate nucleotide channeling in the cell.

Webb PA, Perisic O, Mendola CE, Backer JM, Williams RL
The crystal structure of a human nucleoside diphosphate kinase, NM23-H2.
J Mol Biol. 1995; 251: 574-87
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The 2.8 A resolution X-ray structure of NM23-H2 has been determined by molecular replacement using the structure of Myxococcus xanthus nucleoside diphosphate (NDP) kinase. NM23-H2 is a human NDP kinase. The enzyme catalyses phosphoryl transfer, binds DNA, and can activate the transcription of the c-myc oncogene in vitro. NM23 has also been reported to be a suppressor of metastasis in some types of tumours. Whereas the M. xanthus NDP kinase is a tetramer, NM23-H2 is a hexamer. The fold of NM23-H2 is identical to the fold of other NDP kinases. Two antiparallel helices joined by a turn form one edge of the nucleotide binding cleft. This region moves in a hinge-like fashion in response to substrate binding and crystal packing forces. Additional differences in conformation among the NDP kinases are principally in regions involved in protein-protein contacts within the oligomers. The only protein-protein interaction conserved among all NDP kinases is a dimeric interaction. Several mutations of NM23-H2 have been detected in tumour tissues. These mutations do not involve residues interacting with the substrates, and probably destabilise the enzyme without directly affecting the catalytic activity. Low level phosphorylation of serines has been reported for NM23 both in vitro and in vivo. The structure of the hexamer indicates that two serine residues that have been reported as being phosphorylated, Ser44 and Ser122, are on the surface of the hexamer, and are likely to be phosphorylated by exogenous kinases. In contrast, Ser120 is buried, and is most likely phosphorylated by a direct transfer from the phosphohistidine intermediate of the reaction mechanism.

Morera S, Lacombe ML, Xu Y, LeBras G, Janin J
X-ray structure of human nucleoside diphosphate kinase B complexed with GDP at 2 A resolution.
Structure. 1995; 3: 1307-14
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BACKGROUND: Nucleoside diphosphate (NDP) kinases provide precursors for DNA and RNA synthesis. In mammals, these enzymes are also involved in cell regulations. Human NDP kinase B, product of the human nm23-H2 gene, is both an enzyme and a transcription factor. It activates transcription of the c-myc oncogene independently of its catalytic function, by binding to its promoter DNA. How do the two functions coexist? RESULTS: Recombinant human NDP kinase B was co-crystallized with GDP. The X-ray structure was solved at 2.0 A resolution by molecular replacement from the homologous Drosophila Awd protein. Both enzymes are homo-hexamers with a characteristic beta alpha beta beta alpha beta fold. GDP binds near the active site His118. The guanine base is in a surface cleft and interacts with the C terminus of another subunit. CONCLUSIONS: The beta alpha beta beta alpha beta fold, also present in the 'palm' domain of Escherichia coli DNA polymerase I and HIV reverse transcriptase, is both a mononucleotide- and a polynucleotide-binding fold. If NDP kinase B binds DNA in the same way as the polymerases, the enzyme must undergo a conformation change in order to carry out gene activation.

Jiang Y, He A, Xia S
[Immunohistochemical analysis of nm23 gene product/nucleoside-diphosphate kinase expression in human lung carcinoma]
Zhonghua Jie He He Hu Xi Za Zhi. 1995; 18: 209-11
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It was proposed that nm23 gene may function as a suppressor gene for tumor metastasis. Using an antibody to NDPK, levels of NDPK/nm23 expression in lung carcinomas were examined immunohistochemically, 45 of 58 squamous carcinomas and 26 of 36 adenocarcinomas showed strong immunoreactivity for NDPK/nm23 in most of cancer cells within tumor tissues. On some normal bronchial epithelium and serous glands, there also show weak expression. No relationship was found between NDPK/nm23 expression and clinicopathological parameters including tumor grade, size, nodal involvement and stage but the increases in NDPK/nm23 expression were stronger in advanced stages of squamous cell carcinomas. In conclusion, our results indicate that the increased NDPK/nm23 expression in the analysed carcinomas is not consistent with the proposed metastasis-suppressor function, but the nm23 gene may be implicated in the mechanism of carcinogenic process and tumor progression.

Okabe-Kado J, Kasukabe T, Baba H, Urano T, Shiku H, Honma Y
Inhibitory action of nm23 proteins on induction of erythroid differentiation of human leukemia cells.
Biochim Biophys Acta. 1995; 1267: 101-6
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We recently identified a differentiation inhibitory factor (I-factor) in mouse myeloid leukemia M1 cells as a murine homolog of the human nm23-H2 gene product. nm23 genes encode proteins that participate in tumor metastasis regulation and in various fundamental cellular processes, although their mechanisms of action are still unknown. Although all nm23 proteins contain nucleoside diphosphate (NDP) kinase activity, it has not been established that the enzyme activity mediated the various functions of nm23 proteins. In the present experiment, we examined the effect of nm23 proteins on various differentiation induction systems of human leukemic cells including HL-60, U937, HEL/S, KU812F, K562, and HEL cells. Native human erythrocyte NDP kinase protein inhibited the induction of erythroid differentiation of HEL, KU812 and K562 cells, but not the induction of monocytic or granulocytic differentiation of HL-60, U937 and HEL/S cells. The erythroid differentiation of HEL cells was inhibited by recombinant human nm23-H1, -H2, mouse nm23-M1, and -M2 proteins. Moreover, both the mutant nm23-H2His protein and truncated nm23-H2 protein containing N-terminal (1-60) peptide, which do not have NDP kinase activity, also inhibited erythroid differentiation of HEL cells. These results suggest that (1) the differentiation inhibitory activity of I-factor/nm23 protein is not restricted to monocytic differentiation of M1 cells, (2) the inhibitory activity is exhibited without species specificity, and (3) the differentiation inhibitory activity of the nm23/NDP kinase protein is independent of its enzyme activity and requires the presence of N-terminal peptides.

Timmons L, Xu J, Hersperger G, Deng XF, Shearn A
Point mutations in awdKpn which revert the prune/Killer of prune lethal interaction affect conserved residues that are involved in nucleoside diphosphate kinase substrate binding and catalysis.
J Biol Chem. 1995; 270: 23021-30
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The awd gene of Drosophila melanogaster encodes a nucleoside diphosphate kinase. Killer of prune (Kpn) is a mutation in the awd gene which substitutes Ser for Pro at position 97 and causes dominant lethality in individuals that do not have a functional prune gene. This lethality is not due to an inadequate amount of nucleoside diphosphate (NDP) kinase activity. In order to understand why the prune/Killer of prune combination is lethal, even in the presence of an adequate NDP kinase specific activity level, and to understand the biochemical basis for the conditional lethality of the awdKpn mutation, we generated second site mutations which revert this lethal interaction. All of the 12 revertants we recovered are second site mutations of the awdKpn gene. Three revertants have deletions of the awdKpn protein coding region. Two revertants have substitutions of the initiator methionine and do not accumulate KPN protein. Seven revertants have amino acid substitutions of conserved residues that are likely to affect the active site: five of these have no enzymatic activity and two have a very low level of specific activity. These data suggest that an altered NDP kinase activity is involved in the mechanism underlying the conditional lethality of the awdKpn mutation.

Caligo MA et al.
NM23 gene expression correlates with cell growth rate and S-phase.
Int J Cancer. 1995; 60: 837-42
Display abstract
Two human NM23 genes have been identified: NM23.H1 and NM23.H2 coding for the A and B subunit of a nucleoside diphosphate kinase (NDPK), respectively. NM23.H1 gene has been proposed as a suppressor of metastatic ability in tumor cells, NM23.H2 is identical to the c-myc transcription factor, PuF. The NM23 coding sequence is strongly preserved through different species. Indirect evidence of various types has been accumulated and seems to support an implication of NM23 in cell proliferation. This report shows that the NM23 gene expression is strictly related to the growth state of the cells. Two different in vitro systems (human peripheral blood lymphocytes and human breast epithelial cell line MCF-10A) and one in vivo (human primary infiltrating ductal breast carcinomas) system have been investigated. The mRNA is present in PHA-stimulated peripheral blood lymphocytes, whereas it is nearly undetectable in their resting counterparts. The level of the NM23 gene expression parallels the fraction of cells incorporating thymidine (S-phase) in neoplastic mammary tissues. In synchronously cycling MCF-10A cells NM23.H1 mRNA reaches a maximum abundance in the S-phase and is absent or only present at very low levels during G0/G1 phase, whereas NM23.H2 is present in growth-arrested cells but is upregulated following serum growth stimulation.

Shankar S, Schlictman D, Chakrabarty AM
Regulation of nucleoside diphosphate kinase and an alternative kinase in Escherichia coli: role of the sspA and rnk genes in nucleoside triphosphate formation.
Mol Microbiol. 1995; 17: 935-43
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We have previously reported that two genes cloned from a cosmid library of Escherichia coli can restore mucoidy to an algR2 mutant of Pseudomonas aeruginosa. AlgR2 is a protein involved in the regulation of nucleoside diphosphate kinase (Ndk) as well as alginate synthesis in P. aeruginosa. One of the E. coli genes, rnk, encodes a 14.9 kDa protein with no homology to any other proteins. The other gene, sspA, encodes the stringent starvation protein, a regulatory protein involved in stationary-phase regulation and the stringent response of E. coli. While both rnk and sspA restored alginate production to the P. aeruginosa algR2 mutant, only rnk restored Ndk activity to the mutant. In this report, we have examined the effect of mutations in rnk and sspA on the levels of Ndk in E. coli. We find that a mutation in rnk drastically reduces the level of Ndk in E. coli. A mutation in sspA, however, affects the level of another nucleoside diphosphate kinase distinct from Ndk. The proteins can be easily distinguished from each other by their different affinities for nucleoside diphosphates (NDPs) and also by the differential effect of anti-Ndk antibodies on the reactions they catalyse. The ability of either of these two proteins to restore alginate synthesis in the algR2 mutant of P. aeruginosa demonstrates the importance of nucleoside triphosphate synthesis and energy metabolism for alginate synthesis. Additionally, a role for the stringent starvation protein (SspA) in the modulation of nucleoside triphosphate (NTP) levels in E. coli is also suggested from these experiments.

Ozeki Y, Takishima K, Mamiya G
Immunohistochemical analysis of nm23/NDP kinase expression in human lung adenocarcinoma: association with tumor progression in Clara cell type.
Jpn J Cancer Res. 1994; 85: 840-6
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Levels of nm23-H1/nucleoside diphosphate kinase (NDP kinase) expression have been reported to correlate inversely with metastatic potential in some tumors but not in others. Whether or not nm23 gene product is associated with metastatic potential in lung cancer is not clear as yet. We therefore immunohistochemically examined the expression of nm23 gene products in primary lung adenocarcinomas according to cytologic subtypes in order to clarify the association of its expression with the clinical features of the disease. Seventy-two (64.9%) of the 111 lung adenocarcinomas were positive for nm23 protein. In lung adenocarcinoma of Clara cell type, high levels of nm23 expression were associated with advanced pathologic stage, positive lymph node status, and poorer prognosis (P < 0.05). However, no correlation with clinical outcome was observed in other cell types. Our data suggest that higher levels of nm23 expression are associated with tumor progression in lung adenocarcinoma of Clara cell type.

Bominaar AA, Tepper AD, Veron M
Autophosphorylation of nucleoside diphosphate kinase on non-histidine residues.
FEBS Lett. 1994; 353: 5-8
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Recently, several reports appeared which described auto-phosphorylation of NDP kinase on residues different from the active-site histidine. Based on these findings conclusions were drawn with respect to a regulation of enzyme activity and to a possible role as a metastasis suppressor. In this paper we show that although non-histidine autophosphorylation occurs on NDP kinases from mammals, lower eukaryotes and bacteria, less than 0.2% of the subunits are phosphorylated. Using site-directed mutagenesis, we show that the active site histidine is essential for non-histidine autophosphorylation. The low stoichiometry of phosphate incorporation excludes a role of autophosphorylation in regulating overall enzyme activity.

Tepper AD, Dammann H, Bominaar AA, Veron M
Investigation of the active site and the conformational stability of nucleoside diphosphate kinase by site-directed mutagenesis.
J Biol Chem. 1994; 269: 32175-80
Display abstract
Nucleoside-diphosphate kinase (EC 2.7.4.6) catalyzes phosphate exchange between nucleoside triphosphates and nucleoside diphosphates. Its 17 kDa subunits are highly conserved throughout evolution in both sequence and tertiary structure. Using site-directed mutagenesis we investigated the function of 8 amino acids (Lys16, Tyr56, Arg92, Thr98, Arg109, Asn119, Ser124, and Glu133) that are totally conserved among all nucleoside diphosphate kinases known to date. The mutant proteins all show decreased specific activity and support roles for these residues in catalysis, substrate binding, or both, as was previously proposed on the basis of the x-ray structure (Morera, S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Veron, M., and Janin, J. (1994) Biochemistry 33, 459-467). Furthermore, residues Lys16, Arg109, and Asn 119 were identified to play important roles in conformational stability or subunit interactions. We show that Lys16 and Asn119 form a rigid structure that is important for enzymatic function and that Arg109, known to interact with the phosphate moiety of the substrate, also plays an important role in subunit association. The dual roles of Lys16, Arg109, and Asn119 in both substrate binding and subunit assembly provide further evidence for a functional coupling between catalytic activity and quaternary structure in nucleoside diphosphate kinase.

Veron M et al.
Nucleoside diphosphate kinase: an old enzyme with new functions?
Adv Exp Med Biol. 1994; 370: 607-11

Royds JA, Rees RC, Stephenson TJ
nm23--a metastasis suppressor gene?
J Pathol. 1994; 173: 211-2

Sommer D, Song PS
A plant nucleoside diphosphate kinase homologous to the human Nm23 gene product: purification and characterization.
Biochim Biophys Acta. 1994; 1222: 464-70
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Nucleoside diphosphate kinases (NDPKs) catalyze the transfer of high-energy phosphates from nucleoside triphosphates to nucleoside diphosphates and may be involved in the regulation of growth, development, and signal transduction processes. We report here the purification and characterization of NDPK from detergent-solubilized extracts of dark-grown oat (Avena) tissue. The purification was achieved primarily through adsorption to GTP-agarose, followed by elution with ATP. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography indicated that the purified protein is composed of six 18 kDa subunits. Substrate specificity experiments indicated that the purified kinase is capable of using all tested nucleosides as substrates. N-terminal sequencing of the Avena protein revealed that 87% of the 23 amino acids sequenced were identical to the human Nm23 protein, a nucleoside diphosphate kinase identified as a possible tumor metastasis suppressor and transcriptional activator of the myc oncogene.

Morera S, LeBras G, Lascu I, Lacombe ML, Veron M, Janin J
Refined X-ray structure of Dictyostelium discoideum nucleoside diphosphate kinase at 1.8 A resolution.
J Mol Biol. 1994; 243: 873-90
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The X-ray structure of the nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been refined at 1.8 A resolution from a hexagonal crystal form with a 17 kDa monomer in its asymmetric unit. The atomic model was derived from the previously determined structure of a point mutant of the protein. It contains 150 amino acid residues out of 155, and 95 solvent molecules. The R-factor is 0.196 and the estimated accuracy of the average atomic position, 0.25 A. The Dictyostelium structure is described in detail and compared to those of Drosophila and Myxococcus xanthus NDP kinases. The protein is a hexamer with D3 symmetry. Residues 8 to 138 of each subunit form a globular alpha/beta domain. The four-stranded beta-sheet is antiparallel; its topology is different from other phosphate transfer enzymes, and also from the HPr protein which, like NDP kinase, carries a phosphorylated histidine. The same topology is nevertheless found in several other proteins that bind mononucleotides, RNA or DNA. Strand connections in NDP kinase involve alpha-helices and a 20-residue segment called the Kpn loop. The beta-sheet is regular except for a beta-bulge in edge strand beta 2 and a gamma-turn at residue Ile120 just preceding strand beta 4. The latter may induce strain in the main chain near the active site His122. The alpha 1 beta 2 motif participates in forming dimers within the hexamer, helices alpha 1 and alpha 3, the Kpn loop and C terminus, in forming trimers. The subunit fold and dimer interactions found in Dictyostelium are conserved in other NDP kinases. Trimer interactions probably occur in all eukaryotic enzymes. They are absent in the bacterial Myxococcus xanthus enzyme which is a tetramer, even though the subunit structure is very similar. In Dictyostelium, contacts between Kpn loops near the 3-fold axis block access to a central cavity lined with polar residues and filled with well-defined solvent molecules. Biochemical data on point mutants highlight the contribution of the Kpn loop to protein stability. In Myxococcus, the Kpn loops are on the tetramer surface and their sequence is poorly conserved. Yet, their conformation is maintained and they make a similar contribution to the substrate binding site.

Postel EH, Ferrone CA
Nucleoside diphosphate kinase enzyme activity of NM23-H2/PuF is not required for its DNA binding and in vitro transcriptional functions.
J Biol Chem. 1994; 269: 8627-30
Display abstract
nm23 genes encode proteins that participate in tumor metastasis regulation and in various fundamental cellular processes, although the mechanisms remain undefined. All Nm23 proteins contain nucleoside diphosphate kinase (NDPK) activity whose significance to these regulatory effects is not yet evident. The protein product of the human nm23-H2 gene functions in vitro both as a nucleoside diphosphate kinase enzyme (NDPK-B; Gilles, A.-M., Presecan, E., Vonica, A. and Lascu, I. (1991) J. Biol. Chem. 266, 8784-8789) and as a transcription factor (PuF; Postel, E. H., Berberich, S. J., Flint, S. J. and Ferrone, C. A. (1993) Science 261, 478-480). To understand the significance of these two biochemical activities to NM23-H2 function, we have investigated the relationship between the DNA binding and transcriptional activity of NM23-H2 and its NDPK function. Using site-directed mutagenesis of the cDNA encoding NM23-H2, we have created a mutant substituting for the amino acid histidine 118, the presumed site of phosphorylation in the formation of the phosphoenzyme intermediate, the nonphosphorylatable amino acid phenylalanine. The H118F mutant protein is shown to be catalytically inactive as measured both in a radioisotopic assay that detects formation of the phosphorylated enzyme intermediate and in a coupled enzyme assay that indicates nucleoside diphosphate formation. These results confirm that histidine 118 is the critical residue for NDPK-B activity. In addition, the H118F mutant protein lacking enzymatic activity displayed normal DNA binding affinity for the c-myc promoter in electrophoretic mobility shift assays, and retained full transcriptional activity using the c-myc gene in vitro. These results indicate a lack of correlation between nucleoside diphosphate kinase activity of nm23-H2 on the one hand, and its DNA binding and transcriptional activity on the other, suggesting that the nm23-H2 gene encodes a bifunctional protein molecule.

Kavanaugh-Black A, Connolly DM, Chugani SA, Chakrabarty AM
Characterization of nucleoside-diphosphate kinase from Pseudomonas aeruginosa: complex formation with succinyl-CoA synthetase.
Proc Natl Acad Sci U S A. 1994; 91: 5883-7
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The enzyme nucleoside-diphosphate kinase (Ndk), responsible for the conversion of (deoxy)ribonucleoside diphosphates to their corresponding triphosphates, has been purified from Pseudomonas aeruginosa. The N-terminal 12 amino acid sequence of P. aeruginosa Ndk shows significant homology with that of Myxococcus xanthus and that of Escherichia coli. Ndk enzyme activity is also associated with succinyl-CoA synthetase activity in P. aeruginosa, whose alpha and beta subunits show extensive sequence homology with those of E. coli and Dictyostelium discoideum. The 33-kDa alpha subunit of succinyl-CoA synthetase of P. aeruginosa appears to undergo autophosphorylation in the presence of either ATP or GTP, although the presence of small amounts of Ndk activity may influence the level of such phosphorylation.

Harris N, Taylor JE, Roberts JA
Isolation of a mRNA encoding a nucleoside diphosphate kinase from tomato that is up-regulated by wounding.
Plant Mol Biol. 1994; 25: 739-42
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A cDNA clone (TAB2) encoding a nucleoside diphosphate (NDP) kinase has been isolated from a tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) cDNA library. The clone is 590 bp long and exhibits a high degree of sequence identity with spinach NDP kinases I and II, Pisum sativum NDP kinase I, Arabidopsis thaliana NDP kinase, Drosophila melanogaster NDP kinase, Dictyostelium discoideum NDP kinase and human Nm 23-H1 and Nm23-H2. Northern analysis has revealed that the mRNA encoded by TAB2 is up-regulated in both leaf and stem tissue in response to wounding. The increase is apparent within 1 h of wounding and is not further elevated by application of ethylene. Southern blot analysis indicates that TAB2 is a member of a small gene family.

Moisyadi S, Dharmasiri S, Harrington HM, Lukas TJ
Characterization of a low molecular mass autophosphorylating protein in cultured sugarcane cells and its identification as a nucleoside diphosphate kinase.
Plant Physiol. 1994; 104: 1401-9
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A low molecular mass (18 kD) phosphoprotein (pp18) was characterized and purified from cultured sugarcane (Saccharum officinarum L.) cell line H50-7209. Autophosphorylation assays were used to detect pp18 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only pp18 was detected by a brief in situ phosphorylation method, whereas additional putative protein kinases were detected by an extended method. pp18 was present in both microsomal membrane and soluble fractions and exhibited anomalous turnover of 32P label during in vitro phosphorylation experiments with highest levels present at shorter incubation times. Two major isoforms of the protein were identified in two-dimensional isoelectric focusing/SDS-PAGE of crude extracts and microsomal fractions. The levels of pp18 were enhanced approximately 4-fold by heat shock at 36 degrees C and the elevated pp18 decayed after heat shock was discontinued. pp18 was purified to apparent homogeneity, could be phosphorylated on serine residues, and also exhibited kinase-like activity toward histone H1. The amino acid sequence obtained from a cyanogen bromide digest was greater than 80% identical to nucleoside diphosphate (NDP) kinases from a variety of organisms. Biochemical analysis of the purified protein confirmed the identity as NDP kinase. Thus, NDP kinase appears to be modulated by heat shock in plants.

Swindells MG, Alexandrov NN
Nucleotide binding in beta alpha beta--beta alpha beta topologies.
Nat Struct Biol. 1994; 1: 677-8

Finan PM, White IR, Redpath SH, Findlay JB, Millner PA
Molecular cloning, sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum.
Plant Mol Biol. 1994; 25: 59-67
Display abstract
Protein sequence data derived from the N-terminal region of a 17 kDa polypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were used to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein encoded by NDK-P1 had a calculated molecular mass of 16,485 Da and possessed substantial homology with nucleoside diphosphate kinases (NDKs) isolated and cloned from other sources. High levels of expression of NDK-P1 protein were achieved in Escherichia coli using a T7-driven expression system. Recombinant NDK-P1 protein was shown to possess NDK activity and had similar biochemical characteristics to NDKs isolated from other sources. The Michaelis constants for a variety of nucleoside diphosphate (NDP) substrates were found to be broadly similar to those reported for other NDKs, with thymidine nucleotides being the substrates of greatest affinity.

Chang CL et al.
Nm23-H1 mutation in neuroblastoma.
Nature. 1994; 370: 335-6

Dooley S et al.
Isolation and characterization of the human genomic locus coding for the putative metastasis control gene nm23-H1.
Hum Genet. 1994; 93: 63-6
Display abstract
Nm23-H1 gene expression is inversely correlated with tumor metastatic potential in certain tumors, including melanomas, breast carcinomas, and hepatocellular carcinomas. Using nm23-H1 c-DNA primer and genomic polymerase chain reaction (PCR) amplification, we purified three PCR fragments (one of 4kb and two of 2 kb) covering the whole human genomic locus of the gene (8.460bp). We recombined the PCR products into pUC18 and produced a restriction map to perform subcloning. Complete sequencing of genomic PCR fragments, including the whole coding region of nm23-H1, revealed that the gene consists of five exons and four introns spanning 8.5kb. A sequence homology analysis between human nm23-H1 and the homolog gene of the rat (NDP-K beta) shows that exon-intron boundaries are well conserved between these two species.

Fukuchi T, Shimada N, Hanai N, Ishikawa N, Watanabe K, Kimura N
Recombinant rat nucleoside diphosphate kinase isoforms (alpha and beta): purification, properties and application to immunological detection of native isoforms in rat tissues.
Biochim Biophys Acta. 1994; 1205: 113-22
Display abstract
We previously demonstrated that at least two isoforms of nucleoside diphosphate (NDP) kinase, the products of two different tandemly arrayed genes, are present in rat. To understand the physiological role of each isoform, some biochemical properties of recombinant rat NDP kinase alpha- and beta-isoforms, produced in large amount, were studied. cDNAs of the two isoforms were inserted in an expression vector pET3b and recombinant enzymes were overproduced in Escherichia coli. Their primary structures were different from the native enzymes in that the latter suffer from modification of the NH2-terminal end. The two recombinant isoforms were purified from the cell lysate to apparent homogeneity by ammonium sulfate fractionation followed by three successive column chromatographies. Despite their extreme similarity in the amino-acid sequences, the two showed somewhat different enzymic properties in terms of di- and triphosphate nucleotide substrate specificity. They showed similar mobilities on SDS-PAGE as expected from their calculated molecular weight (alpha-isoform, 17,283 versus beta-isoform, 17,192) but differed in isoelectric point (alpha-isoform, pI 6.7; beta-isoform, pI 7.8) and heat stability. Polyclonal antibody which reacted with both isoforms and alpha-isoform-specific monoclonal antibodies differentially recognized native enzymes from rat tissues after the tissue extracts were separated by isoelectric focusing gel electrophoresis under a denaturation condition. The results showed that the alpha-isoform, though its amount varied from one tissue to another, was the major form in rat tissues examined compared with the beta-isoform which was detectable in brain and testis. There was no preference in their subcellular localization when examined with myelin, synaptosomal supernatant and total homogenate fractions from the rat cerebrum and cerebellum.

Engel M et al.
Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro.
Biochem Biophys Res Commun. 1994; 199: 1041-8
Display abstract
We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia cells were used. The homologous NDPK's from Yeast and E. coli were also substrates for CK-2 in vitro, but not Drosophila NDPK. Phosphorylation of all NDPK types by the CK-2 holoenzyme was entirely polyamine-dependent. The CK-2 phosphorylation site in human NDPK A, that was about 2.5 times stronger phosphorylated than was the B isotype, was tentatively assigned to Ser-122. The location of the corresponding residue in the 3D-structure of the 80% homologous Drosophila NDPK suggests that its phosphorylation may directly influence substrate binding and/or catalysis.

Cherfils J, Morera S, Lascu I, Veron M, Janin J
X-ray structure of nucleoside diphosphate kinase complexed with thymidine diphosphate and Mg2+ at 2-A resolution.
Biochemistry. 1994; 33: 9062-9
Display abstract
We report the crystal structure of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum with thymidine diphosphate (dTDP) and Mg2+ bound at the active site. The structure has been refined to an R-factor of 18.3% at 2-A resolution. The base stacks on the aromatic ring of Phe 64 near the protein surface and is wedged between the side chains of Phe 64 and Val 116. The sugar and the pyrophosphate are deeper inside the protein and make numerous H-bonds with protein side chains. There is no backbone interaction with the nucleotide. A Mg2+ ion bridges the alpha- and beta-phosphates and interacts with the protein via water molecules. NDP kinase shows little specificity toward ribonucleotides and deoxyribonucleotides. This property, required by the enzyme biological function, can now be analyzed by comparing the crystal structures of free, ADP-ligated, and dTDP-ligated enzymes. The most significant differences are located in residues 60-64, which adapt their conformation to allow Phe 64 to stack on both types of bases. Nonspecific binding is achieved by the absence of polar interaction between the base and protein atoms. The ribose of ADP and the deoxyribose of dTDP occupy similar positions, their hydroxyl groups interacting with Lys 16 and Asn 119. The H-bond between Lys 16 and the O2' hydroxyl of ADP is replaced by a similar interaction with a water molecule in the dTDP complex. The beta-phosphate position is the same for ADP and dTDP, suggesting that the mechanism of phosphate transfer is the same for all substrates ofNDP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

MacDonald NJ, De la Rosa A, Benedict MA, Freije JM, Krutsch H, Steeg PS
A serine phosphorylation of Nm23, and not its nucleoside diphosphate kinase activity, correlates with suppression of tumor metastatic potential.
J Biol Chem. 1993; 268: 25780-9
Display abstract
We describe a serine phosphorylation of the putative metastasis suppressor protein Nm23, and present evidence of its relevance to the signal transduction and tumor metastatic processes. Nm23 was previously demonstrated to exhibit nucleoside diphosphate kinase (NDPK) activity, which transfers a phosphate among nucleoside tri- and diphosphates via an Nm23-phospho-histidine intermediate. Recent data have dissociated the NDPK activity of Nm23 from its phenotypic effects; therefore we have asked whether Nm23 possesses additional biochemical functions. An acid-stable (nonhistidine) phosphorylation was identified on autophosphorylated purified recombinant Nm23 proteins and [32P]orthophosphate-labeled human breast carcinoma and murine melanoma Nm23. Phosphoamino acid analysis identified serine as the acid-stable phosphorylation and serine 44 as the major site of phosphorylation. The acid stable phosphorylation (serine) of Nm23 was inhibited by cAMP in vitro and forskolin in vivo, suggesting that this phosphorylation pathway is regulated in signal transduction. No effect of cAMP was observed on Nm23 NDPK activity. Once phosphorylated, Nm23-phosphoserine can release free phosphate in vitro. The biological relevance of the novel phosphorylation identified herein is suggested by the direct correlation of in vivo Nm23 acid-stable phosphorylation levels, but not Nm23 NDPK activity, with suppression of tumor metastatic potential among control and nm23-1 transfected murine melanoma cells.

Zhang J et al.
Nucleotide sequence of the cDNA encoding nucleoside diphosphate kinase II from spinach leaves.
Biochim Biophys Acta. 1993; 1171: 304-6
Display abstract
The primary structure of nucleoside diphosphate (NDP) kinase II, one of the two isozymes found in spinach leaves, has been deduced from its cDNA sequence. NDP kinase II comprises 233 amino acid residues and has a molecular mass of 26,107 Da, which is larger than that of the purified NDP kinase II subunits (18 kDa) by about 8 kDa, suggesting that NDP kinase II might be post-translationally processed. Homology was found between the sequence of spinach NDP kinase II, and the sequences of spinach NDP kinase I, rat NDP kinases alpha and beta, Dictyostelium discoideum NDP kinase, the human Nm23-H1 and Nm23-H2 proteins and the awd protein of Drosophila melanogaster.

Chiadmi M et al.
Crystal structure of the Awd nucleotide diphosphate kinase from Drosophila.
Structure. 1993; 1: 283-93
Display abstract
BACKGROUND: Nucleotide diphosphate kinase (NDP kinase) is a phosphate transfer enzyme involved in cell regulation and in animal development. Drosophila NDP kinase is the product of the abnormal wing disc (awd) developmental gene, a point mutation in which can produce the killer of prune (K-pn) conditional lethal phenotype. The highly homologous mammalian genes control metastasis and a human NDP kinase acts as a transcription factor. RESULTS: The X-ray structure of the Awd protein prepared from Drosophila was solved at 2.4 A resolution by molecular replacement from the homologous Dictyostelium protein. Both are hexamers, and both have the same fold and the same active site. Subunit contacts differ as a result of sequence changes in the carboxy-terminal segment and in the loop that is the site of the K-pn mutation. CONCLUSIONS: Regulatory properties of animal NDP kinases depend on interactions with other macromolecules, such as DNA and the product of the Drosophila prune gene. The Awd structure suggests an allosteric mechanism of action of NDP kinase where DNA is the effector and the protein undergoes a major conformational change, possibly dissociating to dimers.

Kimura N
[Signal transducing systems: G protein mediated signaling systems and NDP kinase]
Tanpakushitsu Kakusan Koso. 1993; 38: 1169-79

Konishi N et al.
Expression of nm23-H1 and nm23-H2 proteins in prostate carcinoma.
Jpn J Cancer Res. 1993; 84: 1050-4
Display abstract
The nm23 gene products/nucleoside diphosphate (NDP) kinase expression in prostate carcinomas and benign hyperplasias was evaluated immunohistochemically. Monoclonal antibodies against nm23-H1 and nm23-H2 proteins were prepared using the corresponding proteins fused with glutathione S-transferase as immunogens. Of the 80 cases of nonmetastatic prostate carcinoma examined, 74% (59/80) and 60% (48/80) were immunoreactive for nm23-H1 or nm23-H2 protein, respectively. Negative staining for nm23-H1 occurred in 83% of metastatic lesions, while 34% were negative for nm23-H2. All primary tumors corresponding to the metastases examined showed positive immunostaining for nm23-H1, indicating an inverse relationship between expression of this protein and metastatic status. nm23-H2 protein was detected in 83% of primary tumors and its expression appeared to be significantly correlated to the degree of histological differentiation. In contrast, all cases of benign prostatic hyperplasia showed elevated levels of both nm23-H1 and nm23-H2 expression. These data suggest that the nm23/NDP kinase may play a role in suppressing the expression of malignant potential in prostate carcinomas.

Lacombe ML
[Nucleoside diphosphate kinase/Nm23 and metastatic potency]
Bull Cancer. 1993; 80: 717-22
Display abstract
The nm23-H1 gene encoding the nucleoside diphosphate (NDP) kinase A has been proposed as a tumor metastasis suppressor. Two important features emerge from published data: 1) an inverse correlation between the metastatic invasion and the level of NDP kinase/Nm23 was observed in melanomas, hepatocellular carcinomas and, in some studies, on breast carcinomas; 2) an overexpression of NDP kinase/Nm23 was observed in several solid tumors as compared to normal surrounding tissues, positively correlated with aggressiveness in the case of neuroblastomas. The level of NDP kinase/Nm23 in tumors appears to be altered in different ways, related or not to the metastatic potential, depending on the tissue of origin. Its evaluation as a prognostic or diagnostic marker of tumor invasion and aggressiveness deserves further study.

Postel EH, Berberich SJ, Flint SJ, Ferrone CA
Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis.
Science. 1993; 261: 478-80
Display abstract
A human gene encoding the c-myc purine-binding transcription factor PuF was identified by screening of a cervical carcinoma cell complementary DNA library with a DNA fragment containing PuF binding sites. The 17-kilodalton bacterially produced PuF was shown to have biological activity and properties similar to that of human PuF. DNA sequence analysis of recombinant PuF revealed perfect identity with the human nm23-H2 nucleoside diphosphate kinase gene, a potential negative regulator of cancer metastasis. These results provide a link between nm23 and the c-myc oncogene and suggest that the nm23 protein can function in vitro in the transcriptional regulation of c-myc expression.

Lascu I, Deville-Bonne D, Glaser P, Veron M
Equilibrium dissociation and unfolding of nucleoside diphosphate kinase from Dictyostelium discoideum. Role of proline 100 in the stability of the hexameric enzyme.
J Biol Chem. 1993; 268: 20268-75
Display abstract
The Killer-of-prune (K-pn) mutation in Drosophila corresponds to a Pro-Ser substitution in nucleoside diphosphate kinase (Lascu, I. Chaffotte, A., Limbourg-Bouchon, B., and Veron, M. (1992) J. Biol. Chem. 267, 12775-12781). We investigated the role of the equivalent proline (Pro100) in the formation and stability of the Dictyostelium nucleoside diphosphate kinase hexamers. Mutations to serine or glycine had only little effect on the properties of the native enzyme. However, the mutant drastically affected the subunit interaction in the hexamer and the ability of the isolated subunits to associate in vitro. While the wild-type hexamer inactivated and unfolded concomitantly at 5-6 M urea, the mutant proteins dissociated to monomers at 0.5-2 M urea and unfolded at 2.5-4 M urea. At intermediate urea concentrations, the unique species present in solution was a folded, partially active monomer as shown by size-exclusion chromatography, UV, fluorescence, and CD spectroscopy. Proline 100 is located in a loop involved in subunits contact. Altered conformation of the loop in P100S and P100S mutants demonstrates its crucial role in subunit assembly. We propose to explain the conditional dominance of the K-pn mutation by the presence of a monomeric form of the enzyme that would have deleterious effects in vivo.

Williams RL, Oren DA, Munoz-Dorado J, Inouye S, Inouye M, Arnold E
Crystal structure of Myxococcus xanthus nucleoside diphosphate kinase and its interaction with a nucleotide substrate at 2.0 A resolution.
J Mol Biol. 1993; 234: 1230-47
Display abstract
The X-ray crystallographic structure of nucleoside diphosphate (NDP) kinase from Myxococcus xanthus has been determined using multiple isomorphous replacement techniques and refined at 2.0 A resolution to a crystallographic R-factor of 0.17. This is the first report of the structure of an enzymatically active NDP kinase and of the enzyme with a bound nucleotide. The structure has been determined in P4(3)2(1)2 and I222 crystal forms. The enzyme monomer consists of a four-stranded antiparallel beta-sheet. The surfaces of the sheet are partially covered with five helical segments. There are two protein molecules in the asymmetric unit of the tetragonal crystal form. They form a dimer with an extensive interface in which 1092 A2 per monomer is buried. The majority of the contact area in the dimer interface is between hydrophobic or aromatic residues. Two dimers are related by a crystallographic 2-fold axis to yield a tetramer. This tetramer is also present in the orthorhombic crystals; however, in this case, the 222 symmetry is entirely crystallographic. Upon tetramer formation, an additional 473 A2 of solvent-accessible surface area from each monomer becomes buried. The interface between dimers in the tetramer is stabilized by salt bridges. Equilibrium sedimentation studies are consistent with the enzyme being a tetramer in solution. The structure of a complex of adenosine diphosphate (ADP) with the enzyme was determined and reveals that most of the nucleotide interactions with the protein are with the pyrophosphate and ribose groups, while the base has no hydrogen bonds with the protein and interacts only by stacking with the side chain of Phe59. The Mg2+ interacts with the pyrophosphate of the ADP and via a solvent molecule with the side chain of the conserved Asp120 residue. The mode of interaction with the nucleotide is novel, with the nucleotide binding at the side of the beta-sheet. The structures of the nucleotide in crystals grown in the presence or absence of Mg2+ are essentially identical. In addition, the phosphotransfer reaction from adenosine triphosphate (ATP) to the enzyme can occur without Mg2+. This suggests that only the second step of the reaction in which the enzyme transfers the phosphate to a nucleoside diphosphate acceptor is significantly catalyzed by the metal.

Munoz-Dorado J, Almaula N, Inouye S, Inouye M
Autophosphorylation of nucleoside diphosphate kinase from Myxococcus xanthus.
J Bacteriol. 1993; 175: 1176-81
Display abstract
The nucleoside diphosphate kinase (NDP kinase) from Myxococcus xanthus has been purified to homogeneity and crystallized (J. Munoz-Dorado, M. Inouye, and S. Inouye, J. Biol. Chem. 265:2702-2706, 1990). In the presence of ATP, the NDP kinase was autophosphorylated. Phosphoamino acid analysis was carried out after acid and base hydrolyses of phosphorylated NDP kinase. It was found that the protein was phosphorylated not only at a histidine residue but also at a serine residue. Replacement of histidine 117 with a glutamine residue completely abolished the autophosphorylation and nucleotide-binding activity of the NDP kinase. Since histidine 117 is the only histidine residue that is conserved in all known NDP kinases so far characterized, the results suggest that the phosphohistidine intermediate is formed at this residue during the transphosphorylation reaction from nucleoside triphosphates to nucleoside diphosphates. Preliminary mutational analysis of putative ATP-binding sites is also presented.

Kimura N
[Nm23/NDP kinase and cancer metastasis]
Gan To Kagaku Ryoho. 1993; 20: 332-7
Display abstract
Recent advances in the research of Nm23/NDP kinase, a tumor metastasis suppressor, are summarized in relation to tumor metastasis. The tumor metastatic potential of cancer cells is associated with decreased levels of Nm23/NDP kinase in animal model systems and human cases including breast cancer. One of the two isoforms seems to be responsible for metastatic processes. Whether this altered expression occurs at the transcriptional level or as a result of allelic deletion of the gene remains to be determined. The possible mechanism by which the Nm23/NDP kinase level regulates metastatic potential of cancer cells are discussed in view of its biochemical properties. The potential importance of the interaction between Nm23/NDP kinase and signal transducing systems is postulated in determining the metastatic potential of cancer cells.

Bominaar AA, Molijn AC, Pestel M, Veron M, Van Haastert PJ
Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.
EMBO J. 1993; 12: 2275-9
Display abstract
Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins.

Troll H et al.
Separate nuclear genes encode cytosolic and mitochondrial nucleoside diphosphate kinase in Dictyostelium discoideum.
J Biol Chem. 1993; 268: 25469-75
Display abstract
We have previously isolated cDNA clones for the gip17 gene encoding the cytosolic nucleoside diphosphate (NDP) kinase from Dictyostelium discoideum, and partial cDNAs for guk, a second member of the NDP kinase gene family (Wallet, V., Mutzel, R., Troll, H., Barzu, O., Wurster, B., Veron, M., and Lacombe, M. L. (1990) J. Natl. Cancer Inst. 80, 1199-1202). We now characterize genomic DNA clones for both NDP kinase genes, and we show that guk defines a nuclear-encoded mitochondrial NDP kinase. Isolated D. discoideum mitochondria contain 3% of the total cellular NDP kinase activity. Antibodies which specifically recognize and inhibit the activity of either cytosolic or mitochondrial NDP kinase unambiguously distinguish between these activities. The nascent mitochondrial NDP kinase contains a presequence of 57 amino acids that is removed during import into the organelle as shown by determination of the NH2 terminus of the mature protein from mitochondria. The genes for mitochondrial and cytosolic NDP kinases contain four and two introns, respectively. The positions of the of the introns in the gene for the cytosolic enzyme match exactly the positions of the second and fourth introns in the coding region of its mitochondrial homologue. From these results we conclude that the isozymes diverged from a common ancestor, and we discuss possible phylogenetic pathways for the evolution of cytosolic and organelle NDP kinases.

Sorscher SM, Steeg P, Feramisco JR, Buckmaster C, Boss GR, Meinkoth J
Microinjection of an nm23 specific antibody inhibits cell division in rat embryo fibroblasts.
Biochem Biophys Res Commun. 1993; 195: 336-45
Display abstract
Expression of the nm23/NDP-K gene correlates with reduced metastasis in some tumors and with increased proliferation in both nontransformed and transformed cells in culture. Decreased nm23/NDP-K expression results in mitotic arrest in neuroblasts of developing Drosophila. In order to better understand the biological role(s) of nm23 in non-transformed cells, an nm23-specific antibody was introduced into rat embryo fibroblasts and effects on DNA synthesis and cell cycle progression were analyzed. Microinjection of the nm23 antibody inhibited cell division with no apparent effect on DNA synthesis. Control experiments revealed that the survival of cells injected with the nm23 antibody was similar to that of control antibody injected cells in the absence of cell division. These results suggest that in mammalian fibroblasts, as in Drosophila, nm23 expression may be necessary for progression through the cell cycle.

Lascu I, LeBlay K, Lacombe ML, Presecan E, Veron M
Assay of nucleoside diphosphate kinase in microtiter plates using a peroxidase-coupled method.
Anal Biochem. 1993; 209: 6-8
Display abstract
A sensitive assay for nucleoside diphosphate kinase which utilizes microtiter plates is described. ATP, formed in the reaction between dGTP and ADP, is trapped by the glycerol kinase reaction. A colored compound is generated by glycerol-3-phosphate oxidase and peroxidase. This assay is useful for testing a large number of samples generated by chromatographic techniques or for screening purposes.

Okabe-Kado J, Kasukabe T, Honma Y, Hayashi M, Henzel WJ, Hozumi M
Identity of a differentiation inhibiting factor for mouse myeloid leukemia cells with NM23/nucleoside diphosphate kinase.
Biochem Biophys Res Commun. 1992; 182: 987-94
Display abstract
Mouse myeloid leukemic line M1 cells can be induced to differentiate into the monocyte/macrophage pathway by various inducers. The induction of differentiation of M1 cells can be inhibited by protein inhibitors termed differentiation inhibiting factors (I-factors) in a cell lysate and conditioned medium of differentiation resistant M1 cells. Production of the I-factor activity in resistant M1 cells is well associated with development of resistance of M1 cells to differentiation inducers. We have now purified one of the I-factors from conditioned medium of differentiation resistant M1 cells. The purified I-factor has a relative molecular mass of approximately 16000-17000 Da (16K I-factor). The amino acid sequence of all fragments of the 16K I-factor we have found are identical with Nm23/nucleoside diphosphate kinase (EC2.7.4.6) protein involved in tumor metastasis. The findings indicate that the I-factor, a candidate suppressor protein for differentiation of leukemic cells, is Nm23/nucleoside diphosphate kinase protein.

Finan PM, White IR, Findlay JB, Millner PA
Identification of nucleoside diphosphate kinase from pea microsomal membranes.
Biochem Soc Trans. 1992; 20: 10-10

Keim D et al.
Proliferation-related expression of p19/nm23 nucleoside diphosphate kinase.
J Clin Invest. 1992; 89: 919-24
Display abstract
High level expression of the nm23-H1 gene, which encodes for a nucleoside diphosphate kinase, has been found to correlate with diminished metastasis in some tumors but not in others. We have previously identified the protein product of the nm23-H1 gene in two-dimensional electrophoretic gels and have designated it p19/nm23. In neuroblastoma, higher levels of p19/nm23, which are associated with amplification of the N-myc oncogene, large tumor mass, and metastasis, were observed in advanced stage tumors compared with limited stage disease. Because of the variable expression of nm23-H1 in different tumors, we have investigated the relationship between amounts of the protein and cell proliferation. The levels of p19/nm23 were compared between resting and mitotically stimulated normal human PBLs and in leukemia cells. The amount of p19/nm23 increased in normal lymphocytes in response to mitotic stimulation and paralleled the increase in DNA synthesis. In leukemia cells obtained from patients with different subtypes of acute leukemia, p19/nm23 levels were also increased relative to resting normal lymphocytes. Treatment of mitotically stimulated lymphocytes with cyclosporin, which inhibits proliferation, blocked the increase in p19/nm23; treatment of the leukemia cell line HL-60 with dimethylsulfoxide, which induces terminal differentiation, resulted in diminished levels of p19/nm23. Our data therefore provide evidence that nm23-H1 expression is related to cell proliferative activity.

Higashiyama M et al.
Immunohistochemical analysis of nm23 gene product/NDP kinase expression in pulmonary adenocarcinoma: lack of prognostic value.
Br J Cancer. 1992; 66: 533-6
Display abstract
Levels of nm23 gene product/nucleoside diphosphate kinase (NDP kinase) expression have been demonstrated to correlate inversely with metastatic potential in several tumours, indicating that this could be a useful tool as a prognostic indicator. Using an antibody to NDP kinase, levels of nm23 gene product/NDP kinase expression in pulmonary adenocarcinoma were examined immunohistochemically. Of 88 patients tested, 39 (44%; Group B) showed strong immunoreactivity for NDP kinase in most of cancer cells within the tumour tissues, while 49 (56%; Group A) contained few or no NDP kinase-positive cancer cells. Nm23 gene product/NDP kinase was expressed independently of clinicopathological factors, and unexpectedly, no correlation of survival rates between both Groups could be demonstrated. Thus, in pulmonary adenocarcinoma, levels of nm23 gene product/NDP kinase expression may lack prognostic value.

Melki R, Lascu I, Carlier MF, Veron M
Nucleoside diphosphate kinase does not directly interact with tubulin nor microtubules.
Biochem Biophys Res Commun. 1992; 187: 65-72
Display abstract
Nucleoside diphosphate kinase has been shown to play a role in proliferation and development. Microtubules have been evoked as a possible target of NDP kinase action; in particular it was proposed that NDP kinase could regulate the cellular pool of polymerizable GTP-tubulin by direct phosphorylation of tubulin bound GDP. We show that this reaction does not occur in vitro and also that NDP kinase does not bind to microtubules both in the presence and absence of MAPs. Thus, any possible physiological effect of NDP kinase on microtubule dynamics is exerted only by modulating the concentrations of free guanine nucleotides in the vicinity of microtubules.

Sastre X
[Nucleoside diphosphate kinase (nm23) as marker of metastatic tumoral disease. Defeated hope?]
Rev Prat. 1992; 42: 973-4

Lacombe ML, Jakobs KH
Nucleoside diphosphate kinases as potential new targets for control of development and cancer.
Trends Pharmacol Sci. 1992; 13: 46-8

Hemmerich S, Pecht I
Oligomeric structure and autophosphorylation of nucleoside diphosphate kinase from rat mucosal mast cells.
Biochemistry. 1992; 31: 4580-7
Display abstract
Nucleoside diphosphate (NDP) kinases have been found to be involved in a wide range of fundamental biological processes ranging from developmental control to signal transduction and metastasis. We have recently cloned and sequenced a cDNA encoding an NDP-kinase of the rat mucosal mast cell line RBL-2H3 [Hemmerich, S., Yarden, Y., & Pecht, I. (1992) Biochemistry (preceding paper in this issue)]. The enzyme itself has been isolated by means of its affinity to the bischromone cromoglycate. Here we report several of its biochemical characteristics: A structural model for the native protein is proposed in which two disulfide-linked pairs of similar 18-kDa subunits (p18) associate to form a 72-kDa tetramer (p72). This is based on the migration properties of the purified enzyme on gel filtration columns, sodium dodecylsulfate gel electrophoresis, and two-dimensional electrophoresis, together with peptide mapping data. In the absence of NDP, both intact p72 and the dissociated 18-kDa subunits (p18) were shown to undergo Mg(2+)-dependent stoichiometric autophosphorylation utilizing adenosine and guanosine triphosphate or gamma-thiotriphosphate as phosphate donor. This autophosphorylation activity was found to be retained by the 18-kDa subunits even following fractionation by SDS-PAGE and electrophoretic transfer to nitrocellulose. The Michaelis constant of this autophosphorylation reaction with either ATP, ATP gamma S, GTP, or GTP gamma S was determined to be 6.5 +/- 1 microM, and maximally 2 mol of phosphate were found to be incorporated per p72 molecule, thus indicating that phosphorylation occurs at a single site on only two of the four 18-kDa subunits of the holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemmerich S, Yarden Y, Pecht I
A cromoglycate binding protein from rat mast cells of a leukemia line is a nucleoside diphosphate kinase.
Biochemistry. 1992; 31: 4574-9
Display abstract
Recently, we have shown that a membrane-permeant derivative of the antiasthmatic drug cromoglycate (CG) effectively inhibits the Fc epsilon-receptor-mediated secretory response of rat mucosal mast cells (line RBL-2H3) at a stage preceding the transient rise in the cytoplasmic free calcium concentration [Hemmerich, S., Sijpkins, D., & Pecht, I. (1991) Biochemistry 30, 1523-1532]. In contrast to cromoglycate itself, which is membrane impermeant and ineffective in these cells, its bis-acetoxymethyl ester derivative (CG/AM) can diffuse across the plasma membrane into the cytosol, where it is hydrolyzed into the impermeant CG dianion, which presumably may interact with intracellular components involved in the Fc epsilon R signal transduction pathway. In order to identify cytosolic components involved in the stimulus-secretion coupling that interact with this drug, we coupled CG to an insoluble matrix. This matrix was indeed effective in the affinity isolation from RBL cells of a cytosolic protein that exhibits an apparent molecular mass of 18 kDa on reducing SDS gels. This protein was purified to homogeneity and then fragmented, and the amino acid sequence of three resultant peptides was determined. Using the corresponding synthetic oligonucleotides, we cloned and sequenced a cDNA that encodes the full-length 18-kDa polypeptide (p18). The protein sequence deduced from this cDNA is identical to that of rat nucleoside diphosphate kinase [Kimura, N., Shimada, N., Nomura, K., & Watanabe, K. (1990) J. Biol. Chem. 265, 15744-15749] and highly homologous (88%) to the human NM23 gene product whose expression is associated with reduced metastatic potential, as well as with the Drosophila awd gene product (77% sequence identity).(ABSTRACT TRUNCATED AT 250 WORDS)

Litwin CM, Gendreau M, Wang JH
p56lyn catalyzes a reversible autophosphorylation reaction and a nucleoside diphosphate kinase reaction.
FEBS Lett. 1992; 309: 275-8
Display abstract
The reversible autophosphorylation of the pp60c-src family tyrosine kinase, p56lyn has been characterized by a simple procedure that involves the examination of the enzyme catalyzed radioisotope exchange between ATP and ADP. The equilibrium constant of the reaction was determined to be 3.31 and corresponded to a standard free energy of hydrolysis of the phosphotyrosine bond in p56lyn of -8.08 kcal/mol. GDP was capable of substituting for ADP as phosphate acceptor so that p56lyn displayed a nucleoside diphosphate kinase activity.

Golden A et al.
Nucleoside diphosphate kinases, nm23, and tumor metastasis: possible biochemical mechanisms.
Cancer Treat Res. 1992; 63: 345-58

Randazzo PA, Kahn RA, Northup JK
Nucleoside diphosphate kinase: conclusions withdrawn.
Science. 1992; 257: 862-862

Nakayama T et al.
Expression in human hepatocellular carcinoma of nucleoside diphosphate kinase, a homologue of the nm23 gene product.
J Natl Cancer Inst. 1992; 84: 1349-54
Display abstract
BACKGROUND: Expression of nucleoside diphosphate (NDP) kinase, which is highly homologous to the nm23 gene product in a variety of species, has been found to be inversely associated with metastatic potential in human breast cancer. PURPOSE: The present study was conducted to clarify the association of NDP kinase expression with metastatic potential in human hepatocellular carcinoma. METHODS: The immunohistochemical expression of NDP kinase was analyzed in 30 patients with histopathologically proven hepatocellular carcinoma. These patients included nine with distant metastases and 21 without distant metastases. Tissue specimens were reacted with rabbit anti-rat NDP kinase antibody and stained by the biotin-streptavidin complex method. The relative staining intensities were evaluated by comparing primary tumor sites with adjacent nontumorous liver tissue or with metastatic sites, RESULTS: The expression of NDP kinase in primary sites in patients with distant metastases was significantly less intense than that in patients without distant metastases (P = .018). NDP kinase was expressed significantly less intensely in metastatic sites than in primary sites (P = .005). The intensity of NDP kinase expression did not statistically correlate with tumor size or number of lesions in the liver, histopathological classification of tumor, associated liver diseases, hepatitis virus markers, or tumor markers. CONCLUSION: These results suggest that the reduced expression of NDP kinase is closely associated with distant metastatic potential in hepatocellular carcinoma. IMPLICATIONS: It is possible that both NDP kinase and the nm23 gene product may be active in the progression and differentiation of tumor cells and that their reduced expression induces a high metastatic potential in tumor cells. Studies using Northern blotting or in situ hybridization should be planned to confirm our findings.

Urano T, Takamiya K, Furukawa K, Shiku H
Molecular cloning and functional expression of the second mouse nm23/NDP kinase gene, nm23-M2.
FEBS Lett. 1992; 309: 358-62
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A new murine cDNA of nm23/NDP kinase was isolated. A RT-PCR product was obtained from the normal mouse liver mRNA with primers designed for the human nm23-H2 gene. The product was used as a probe to screen a cDNA library from the murine melanoma cell line, B16, and two clones containing the entire open reading frame were obtained. It was predicted that the DNA sequence encoded 152 amino acids which was 98% identical to the nm23-H2 protein. The entire nm23-M1 and -M2 gene-coding regions were translated as fusion proteins with a glutathione S-transferase. These fusion proteins displayed NDP kinase activities.

Zhang D, Chang KJ
The purification of nucleoside diphosphate kinase from rat brain.
Chin Med Sci J. 1992; 7: 152-6
Display abstract
Nucleoside diphosphate kinase from the cytosol of rat brain was purified to electrophoretic homogeneity through a series of chromatography columns, including DE-52, AcA-34 Ultrogel, phenyl-Sepharose and Q-Sepharose. The molecular weight of the enzyme was 64,000 as estimated by gel filtration, and it consisted of 3 identical 19,000 subunits. [35S]-GTP gamma S binding and autophosphorylation of the enzyme, and inhibition of the GTPase activity of G-protein were used to screen for the enzyme. These methods were shown to be useful for purifying and assaying the enzyme.

Lascu I, Chaffotte A, Limbourg-Bouchon B, Veron M
A Pro/Ser substitution in nucleoside diphosphate kinase of Drosophila melanogaster (mutation killer of prune) affects stability but not catalytic efficiency of the enzyme.
J Biol Chem. 1992; 267: 12775-81
Display abstract
Nucleoside diphosphate kinase of Drosophila, recently identified as the product of the awd gene, is essential for larval development. The conditional lethal mutation Killer of prune maps to the same gene. We purified the nucleoside diphosphate kinases from wild-type and mutant larvae by a simple procedure involving affinity chromatography on blue Sepharose. Both proteins are purified as hexamers in their native state. The mutant protein, which carries a serine instead of proline at position 97, has structural properties and catalytic efficiency that are very similar to the wild-type protein. However, the mutant protein has a much lower stability to denaturation by heat and urea. Following dilution of urea with buffer the urea-denaturated mutant nucleoside diphosphate kinase accumulates as folded monomers and cannot recover its quaternary structure and enzymatic activity. In contrast, the wild-type enzyme recovers hexameric structure and activity. This suggests that the mutation affects the folding/assembly pathway without affecting the function of the mature protein once folded and assembled into the mature hexameric structure.

Lacombe ML, Sastre X, Lascu I, Veron M
[Nucleoside diphosphate kinase (NM23) and cancer]
Bull Cancer. 1991; 78: 583-5

Lacombe ML et al.
Overexpression of nucleoside diphosphate kinase (Nm23) in solid tumours.
Eur J Cancer. 1991; 27: 1302-7
Display abstract
The product of the nm23-H1 gene, reported to be related to the metastatic potential of tumour cells, was recently identified as the nucleoside diphosphate (NDP) kinase A (Gilles et al., 1991, J Biol Chem, 266, 8784-8789). An analysis of the enzyme by activity measurement and immunological techniques using polyclonal antibodies raised against the NDP kinase A purified from human erythrocytes, was performed on 39 human tissue specimens. Markedly increased activity and higher level of the protein were observed in extracts of solid tumours as compared to the corresponding normal tissues (P less than 0.01). An intense immunolabelling of tumoral cells was observed in sections of the malignant tumours and of some but not all benign neoplasia. The staining is observed in noninvasive and invasive ductal breast carcinomas with or without lymph node involvement as well as in colon and cervix carcinomas and in a case of metastatic melanoma. Therefore, NDP kinase A level is increased in neoplastic tissues but no correlation with metastatic potential could be demonstrated.

Gilles AM, Presecan E, Vonica A, Lascu I
Nucleoside diphosphate kinase from human erythrocytes. Structural characterization of the two polypeptide chains responsible for heterogeneity of the hexameric enzyme.
J Biol Chem. 1991; 266: 8784-9
Display abstract
Human erythrocyte nucleoside-diphosphate kinase (NDP kinase) is a hexameric enzyme consisting of two kinds of polypeptide chains, A and B. By random association (A6, A5B...AB5, B6) these polypeptides form isoenzymes differing in their isoelectric point. Chains A and B of NDP kinase were purified by ion-exchange chromatography under denaturing conditions. Upon mixing and renaturation, the isozymic pattern of NDP kinase obtained by conventional methods was restored. Antibodies raised against purified chains showed significant cross-reactivity, both in immunoblot experiments and activity inhibition studies. Sequence determination showed that both chains consisted of 152 amino acid residues corresponding to Mr or 17,143 (chain A) and 17,294 (chain B), respectively. There was high homology between the two sequences (88% identity). The phosphorylation site on the enzyme is located at His-118. Chain A was identical with human Nm23 protein, which has been reported as a potential suppressor protein in tumor metastasis and chain B was identical with Nm23-H2 protein.

Kimura N
[A candidate tumor metastasis suppressor Nm23 and NDP kinase]
Seikagaku. 1991; 63: 33-8

Hama H, Almaula N, Lerner CG, Inouye S, Inouye M
Nucleoside diphosphate kinase from Escherichia coli; its overproduction and sequence comparison with eukaryotic enzymes.
Gene. 1991; 105: 31-6
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The gene encoding nucleoside diphosphate (NDP) kinase of Escherichia coli was identified by polymerase chain reaction using oligodeoxyribonucleotide primers synthesized on the basis of consensus sequences from Myxococcus xanthus and various eukaryotic NDP kinases. The gene (ndk), mapped at 54.2 min on the E. coli chromosome, was cloned and sequenced. The E. coli NDP kinase was found to consist of 143 amino acid residues that are 57, 45, 45, 42, 43, and 43% identical to the M. xanthus, Dictyostelium discoideum, Drosophila melanogaster, mouse, rat, and human enzymes, respectively. The ndk gene appears to be in a monocistronic operon and, when cloned in a pUC vector, NDP kinase was overproduced at a level of approx. 25% of total cellular proteins. The protein could be labeled with [gamma-32P]ATP and migrated at a 16.5 kDa when electrophoresed in SDS-polyacrylamide gel, which is in good agreement with the Mr of the purified E. coli NDP kinase previously reported.

Nomura T, Fukui T, Ichikawa A
Purification and characterization of nucleoside diphosphate kinase from spinach leaves.
Biochim Biophys Acta. 1991; 1077: 47-55
Display abstract
Two types of nucleoside diphosphate kinase (NDP kinase I and NDP kinase II) have been purified from spinach leaves to electrophoretic homogeneity. The enzymes were copurified with apparent [35S]GTP-gamma S-binding activities. NDP kinase I, which was not adsorbed to a hydroxyapatite column, and NDP kinase II, which was adsorbed, had molecular weights of 16,000 and 18,000, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weights determined by gel filtration were 92,000 and 110,000, respectively, suggesting that both enzymes are composed of six identical subunits. Minor differences in some amino acids between NDP kinase I and NDP kinase II were observed when both enzymes were analyzed for amino acid composition. The apparent [35S]GTP gamma S-binding activity of purified NDP kinase I and NDP kinase II was found to be due to the formation of a [35S]thiophosphorylated enzyme, which is the intermediate of the NDP kinase reaction.

Dumas C, Lebras G, Wallet V, Lacombe ML, Veron M, Janin J
Crystallization and preliminary X-ray diffraction studies of nucleoside diphosphate kinase from Dictyostelium discoideum.
J Mol Biol. 1991; 217: 239-40
Display abstract
Nucleoside diphosphate kinase from the slime mold Dictyostelium discoideum is highly homologous to gene products that are involved in development in Drosophila and in oncogenesis in human cells. The cloned protein expressed in Escherichia coli has been purified and crystallized in a hexagonal space group with a = b = 74.9 A, c = 211.4 A. The asymmetric unit contains either one or two 17,000 Mr subunits of the hexamer.

Williams RL, Munoz-Dorado J, Jacobo-Molina A, Inouye S, Inouye M, Arnold E
Crystallization and preliminary X-ray diffraction analysis of oucleoside diphosphate kinase from Myxococcus xanthus.
J Mol Biol. 1991; 220: 5-7
Display abstract
Nucleoside diphosphate (NDP) kinase catalyzes the transfer of the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. Human and rodent forms of this enzyme have been shown to be suppressors of metastasis. Crystals that diffract X-rays to high resolution have been obtained for the recombinant Myxococcus xanthus NDP kinase expressed in and purified from Escherichia coli. Two crystal forms have been obtained. Both forms are orthorhombic, space group I222 (or I2(1)2(1)2(1)) with a = 267.1 A, b = 74.0 A and c = 75.1 A for form I and a = 53.5 A, b = 74.0 A and c = 75.1 A for form II. Form I appears to have five molecules in the asymmetric unit approximately related to each other by a translation of 0.2 along the a axis. Diffraction data have been recorded to 1.9 A for form I and to 2.2 A for form II.

Biggs J, Hersperger E, Steeg PS, Liotta LA, Shearn A
A Drosophila gene that is homologous to a mammalian gene associated with tumor metastasis codes for a nucleoside diphosphate kinase.
Cell. 1990; 63: 933-40
Display abstract
The product of the abnormal wing discs (awd) gene of Drosophila is 78% identical to the product of the nm23 gene of mammals, which is differentially expressed in certain metastatic tumors. We present evidence that the awd gene codes for a nucleoside diphosphate kinase (NDP kinase) and that this Awd/NDP kinase is microtubule associated. Neuroblasts in Drosophila larvae homozygous for a null mutation in the awd gene are arrested in metaphase, indicating that microtubule-associated Awd/NDP kinase plays a critical role in spindle microtubule polymerization.

Lacombe ML, Wallet V, Troll H, Veron M
Functional cloning of a nucleoside diphosphate kinase from Dictyostelium discoideum.
J Biol Chem. 1990; 265: 10012-8
Display abstract
A lambda gt11 cDNA library from Dictyostelium discoideum was screened by direct labeling of filter replicas with [35S]guanosine 5'-O-(thiotriphosphate) (GTP gamma S). A positive clone was obtained and used as probe to isolate additional clones from which a complete cDNA sequence was determined. The cDNA hybridizes to a single copy gene that is expressed as a 0.6-kilobase mRNA in vegetatively growing amoeba. The open reading frame encodes a protein of 155 amino acids (calculated Mr 16,775), devoid of cysteine residues. The protein contains most of the short consensus motifs characteristic of the catalytic domain of protein kinases although the overall homology with this class of enzymes is not greater than 20%. Its size and amino acid composition indicated that it could be the monomer of a nucleoside diphosphate (NDP) kinase, an enzyme which catalyzes the phosphate transfer from triphospho- to diphosphonucleotides. Indeed, specific NDP kinase activity was found in extracts of bacteria transformed with a plasmid expressing the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracts incubated in the presence of [35S]GTP gamma S revealed a single 35S-labeled band of size corresponding to the protein, which likely represents the stable thiophosphorylated reaction intermediate characteristic for the ping-pong reaction mechanism of NDP kinases. The formation of this labeled intermediate probably allowed the detection of the enzyme on the filters during the screening procedure. Although NDP kinases from a great variety of sources have been characterized, the primary structure of the D. discoideum NDP kinase is the first reported for an enzyme of eukaryotic origin.

Wallet V et al.
Dictyostelium nucleoside diphosphate kinase highly homologous to Nm23 and Awd proteins involved in mammalian tumor metastasis and Drosophila development.
J Natl Cancer Inst. 1990; 82: 1199-202
Display abstract
Two complementary DNAs (cDNAs) previously isolated, one by functional screening and the other by immunological screening of a Dictyostelium discoideum expression library, encode two proteins, Gip17 and Guk7.2, sharing 71% homology. In the present study, we found that the expression of their messenger RNAs (mRNAs) is developmentally regulated, with a sharp decrease during the first hours of differentiation. The Gip17 protein was purified to homogeneity from D. discoideum amoebas and from recombinant Escherichia coli and was conclusively identified as a nucleoside diphosphate (NDP) kinase. NDP kinases play a major role in synthesis of nucleoside triphosphates and, in many systems, are found associated with guanosine triphosphate (GTP)-binding proteins. We found the Gip17 protein to be 77% homologous to the human Nm23 protein and 75% homologous to the Drosophila melanogaster Awd protein. The levels of murine and human nm23 mRNA and Nm23 protein are significantly reduced in tumor cells of high metastatic potential, suggesting that Nm23 is involved in suppression of mammalian tumor metastasis, and mutants of the awd gene exhibit widespread development abnormalities, suggesting that Awd is involved in D. melanogaster development. The high percentage of homology of the Gip17 and Guk7.2 proteins with the Nm23 and Awd proteins indicates that Nm23 and Awd also have nucleoside diphosphate kinase activity. Possible modulations in the activity of this metabolic enzyme could be related to the altered metabolism of tumor cells and the control of metastatic potential. Our results point to an unexpected role of NDP kinase in development, growth control, and oncogenic transformation.

Presecan E, Vonica A, Lascu I
Nucleoside diphosphate kinase from human erythrocytes: purification, molecular mass and subunit structure.
FEBS Lett. 1989; 250: 629-32
Display abstract
A new procedure for the purification of nucleoside diphosphate kinase from human erythrocytes is described. The enzyme (105 kDa by gel filtration) is made-up of two different kinds of subunits (19.0 and 20.5 kDa), both displaying enzymatic activity. The probable subunit structure of the enzyme is hexameric. The discrepancies related to earlier work are discussed.

Francis B, Overmeyer J, John W, Marshall E, Haley B
Prevalence of nucleoside diphosphate kinase autophosphorylation in human colon carcinoma versus normal colon homogenates.
Mol Carcinog. 1989; 2: 168-78
Display abstract
The G-regulatory proteins of adenylate cyclase, tubulin, and the ras oncogene protein product require the production of GTP from ATP in order to exert their effects within the cell. This implies that the activity of nucleoside diphosphate kinase (NDPK) plays a major role in the regulation of cellular events requiring GTP and that the level of activity of this enzyme is critical. This report presents a simple method for trapping a specific isozyme of NDPK in its high-energy phosphorylated form (NDPK approximately P) using EDTA and demonstrates that this NDPK approximately P is tenfold higher in malignant colon tumor tissue than in normal colon tissue. This autophosphorylation of the 21,000 and 24,000 Mr subunits of NDPK occurs rapidly at 0 degrees C, will use either [gamma-32P]ATP, [gamma-32P]GTP, or the corresponding 8-azidopurine photoprobes, is intramolecular, displays saturation effects, and is prevented from forming if GTP gamma S is added. Dephosphorylation in the homogenate occurs rapidly upon addition of Mg2+ or any nucleoside-5'-diphosphate. The subunits autophosphorylated in the homogenates are mostly in the soluble phase, and they comigrate with the subunits of pure NDPK from human erythrocytes. Cross-addition of normal and malignant homogenates does not decrease the level of autophosphorylation of NDPK, which indicates that the level of NDPK approximately P may be a quantitative measure of the level of this specific NDPK isozyme form. Assays for NDPK activity show correspondingly elevated levels in the malignant homogenates. Using western blot and photoaffinity labeling techniques, we distinguished the NDPK approximately P subunits from two closely migrating GTP-binding proteins. These were identified as the ras gene protein product and a 20,000 Mr protein, which comigrates identically with ADP-ribosylating factor (ARF). The ARF also comigrates in a tight band that is phosphorylated by [gamma 32P]ATP or [gamma-32P]GTP when Mg2+ is present.

Kimura N
[Possible involvement of nucleotide diphosphate kinase in membrane signal transduction]
Seikagaku. 1987; 59: 1227-33

Ohtsuki K, Yokoyama M
[A possible physiological role of NDP-kinase in cell differentiation and cell proliferation]
Seikagaku. 1986; 58: 1156-63

Islam K, Burns RG
Microtubules and nucleoside diphosphate kinase. Nucleoside diphosphate kinase binds to co-purifying contaminants rather than to microtubule proteins.
Biochem J. 1985; 232: 651-6
Display abstract
Nucleoside diphosphate (NDP) kinase has been postulated to generate GTP from the GDP bound to tubulin. The purified chick brain enzyme was studied with respect to its kinetic parameters, and the protein-protein interactions between the NDP kinase and tubulin were examined. No specific interaction is observed between the enzyme and assembled microtubules, tubulin dimers, or tubulin-microtubule-associated protein (MAP) oligomers under a variety of nucleotide conditions. The apparent association is demonstrated to result from NDP kinase binding to a co-purifying contaminant. The absence of detectable NDP kinase-tubulin interactions indicates that NDP kinase does not directly charge up tubulin-GDP.

Nickerson JA, Wells WW
The microtubule-associated nucleoside diphosphate kinase.
J Biol Chem. 1984; 259: 11297-304
Display abstract
Microtubule protein prepared by cycles of assembly-disassembly contains a nucleoside diphosphate kinase (NDP kinase) activity. We have isolated the NDP kinase responsible for this activity from twice-polymerized bovine brain microtubule protein by a five-step chromatographic procedure. The molecular weight of this enzyme was 103,000 +/- 7,000 daltons as determined by sedimentation equilibrium experiments performed with a Beckman Airfuge. A doublet of subunit bands with molecular masses of about 18,000 daltons was detected by silver staining after gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this preparation. We conclude that the enzyme is a hexamer, although we cannot identify the mix of subunits. We were able to isolate only nanogram quantities of this enzyme, too little for extensive studies, so we isolated the enzyme directly from bovine brain without a preliminary microtubule protein isolation. The whole-brain NDP kinase was isolated by the same chromatographic steps as the enzyme from microtubule protein preparations. Both enzymes had a doublet of subunits at the same molecular weights and both were the same isozyme, chromatofocusing at a pH of 8.0. Both enzymes had similar kinetic properties and similar thermal inactivation profiles. These similar properties of the two enzymes suggest that they are identical. Both subunits of NDP kinase could be reversibly phosphorylated by ATP. Phosphorylation of the native enzyme created multiple, more acidic forms that retained activity. The isolation of this NDP kinase, which can copurify with microtubule protein through cycles of assembly-disassembly, will facilitate future studies on the role of this enzyme in the mechanism and regulation of microtubule assembly.

Savitskii IV, Nagiev ER
[Nucleoside mono- and nucleoside diphosphate kinase activities in rat liver and brain in radiation sickness]
Ukr Biokhim Zh. 1983; 55: 456-9
Display abstract
Activity of nucleoside di- and nucleoside triphosphates metabolism enzymes in tissues of rats gamma-irradiated by a dose of 30 Gy was studied 0.5, 1, 3, 6 and 24 hours after the radiation effect. It is shown that the nucleoside monophosphate kinase activity of the liver and brain is enhanced almost at all stages of the studies and the nucleoside diphosphate kinase activity is inhibited. A significant but reversible decrease of the nucleoside monophosphate kinase activity is observed in the liver 3 h later. By an end of the first day after irradiation the nucleoside mono- and nucleoside diphosphate kinase activities increase significantly both in the liver and brain.

Edlund B
Effects of chemical modification of lysine, tyrosine and tryptophan residues in pea seed nucleoside diphosphate kinase and inhibition of the enzyme with antibodies.
Ups J Med Sci. 1982; 87: 251-8
Display abstract
Pea seed nucleoside diphosphate kinase (NDP kinase) is an oligomeric, tetrameric enzyme which has been shown to be phosphorylated by its substrate ATP, presumably forming an 1-phosphohistidine at its active site. Recently has also a reactive lysine residue been demonstrated at the active site. In the present investigation nitration of a tyrosine residue is shown to inactivate the enzyme. There seems only to be a slight structural change in the enzyme on modification of these essential lysine and tyrosine residues, since double diffusion experiments with an inhibitory antiserum shows no difference in reactivity between the native enzyme and the modified enzyme. It is also found that modification of all tryptophan residues in the enzyme reduces the enzyme activity only to a small degree, indication that these hydrophobic amino acid residues are not directly involved in the catalytic process.

Edlund B, Heldin CH, Engstrom L
Effect of chemical modification of a histidine and a lysine residue of pea seed nucleoside diphosphate kinase.
Ups J Med Sci. 1982; 87: 243-50
Display abstract
Chemical modification of a histidine and lysine residue inactivates pea seed nucleoside diphosphate kinase (NDP kinase). Thus there seems to be a reactive lysine residue, at the active site of pea seed NDP kinase, in addition to the histidine residue phosphorylated by the substrate ATP as a consequence of the enzyme reaction. The presence of a reactive lysine at the active site of the enzyme could explain why a small amount of N-epsilon-phospholysine, as well as 1-phosphohistidine and 3-phosphohistidine, is formed on alkaline hydrolysis of the enzyme.

Dancea S, Dronca M, Ionescu NG, Dimoftache E, Jebeleanu L, Barzu O
Distribution and properties of human liver nucleoside diphosphate kinase.
Int J Biochem. 1981; 13: 609-13

Burns RG, Islam K
Nucleosidediphosphate kinase associates with rings but not with assembled microtubules.
Eur J Biochem. 1981; 117: 515-9
Display abstract
Microtubules reassembled in vitro from chick brain contain significant nucleosidediphosphate (NDP) kinase activity (EC 2.7.4.6) although the specific activity decreases with successive cycles of reassembly. However, while the recovery of microtubule protein, as a function of initial protein concentration, exhibits a critical concentration below which there is no polymerisation, the recovery of NDP kinase activity is, at low initial protein concentrations, directly proportional to the initial protein content indicating that microtubule protein and the kinase activity are independently recovered. This was confirmed by pelleting the reassembled microtubules through a sucrose cushion: the specific activity of the pelleted microtubules was reduced by approximately 90%. By contrast, when cold-dissociated microtubule protein, which is predominantly in the form of rings, is fractionated on a Biogel A 15 m column the microtubule protein and NDP kinase activity coeluted in the void volume and the specific activity remained constant throughout the ring fraction. Similarly, when microtubules were dissociated in the presence of NDP kinase the enzyme bound to the generated rings. These results suggest that NDP kinase binds preferentially to the rings compared with the microtubules, and a model is proposed to account for the presence of this enzyme in pellets of microtubule protein.

Minh Duc D, Lascu I, Porumb H, Gozia O, Schell HD, Barzu O
Differential sensitivity to p-chloromercuribenzoate and urea of soluble and Sepharose-bound pig heart nucleoside diphosphate kinase.
FEBS Lett. 1981; 127: 281-4

Lascu I, Duc M, Cristea A
Rapid large-scale purification of pig heart nucleoside diphosphate kinase by affinity chromatography on Cibacron Blue 3G-A Sepharose.
Anal Biochem. 1981; 113: 207-11

Katz M, Westley J
Enzymic memory studies with nucleoside-5'-diphosphate kinase.
Arch Biochem Biophys. 1980; 204: 464-70

Jacobs M, Huitorel P
Tubulin-associated nucleoside diphosphokinase.
Eur J Biochem. 1979; 99: 613-22
Display abstract
Microtubule protein, prepared by cycles of polymerisation and dissociation, contained a nucleoside diphosphokinase (NDP kinase) activity (EC 2.7.4.6). This activity was not intrinsic to the tubulin dimer or the so-called microtubule-associated proteins. The NDP kinase had the following properties. (1) The enzyme existed in a low-molecular-weight form and in association with the complex of microtubule-associated proteins and tubulin (i.e. multimeric tubulin). (2) The low-molecular-weight species was also formed by dissociation of multimeric tubulin by salt or by removal of microtubule-associated proteins on phosphocellulose. (3) GDP bound to the exchangeable site of multimeric tubulin and also GDP derived from the E site of the tubulin dimer was a substrate for the NDP kinase. (4) The NDP kinase showed a 7-fold increase in activity during ATP-dependent microtubule assembly. On the basis of these properties, it is proposed that microtubule protein contains an NDP kinase specifically associated with tubulin and its functions.

Richard JM
Nucleoside diphosphate kinase d'erythrocytes de boeuf. Caracteristiques cinetiques.
Biochimie. 1977; 59: 665-71

von Dobeln U
Nucleoside diphosphate kinase activity associated with ribonucleotide reductase.
Biochem Biophys Res Commun. 1976; 72: 1160-8