Secondary literature sources for PA2c
The following references were automatically generated.
- Bonfim VL et al.
- Isolation and enzymatic characterization of a basic phospholipase A2 from Bothrops jararacussu snake venom.
- J Protein Chem. 2001; 20: 239-45
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A novel basic phospholipase A2 (PLA2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY...) showed a high degree of homology with basic D49 PLA2 myotoxins from other Bothrops venoms. Bj IV had high PLA2 activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45 degrees C. Full PLA2 activity required Ca2+ but was inhibited by Cu2+ and Zn2+, and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA2 was similar to that in the basic PLA2 of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA2 could partly explain the low PLA2 activity of Bothrops venoms.
- Beghini DG, Toyama MH, Hyslop S, Sodek LC, Novello, Marangoni S
- Enzymatic characterization of a novel phospholipase A2 from Crotalus durissus cascavella rattlesnake (Maracamboia) venom.
- J Protein Chem. 2000; 19: 679-84
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The PLA2 and crotapotin subunits of crotoxin from Crotalus durissus cascavella venom were purified by a combination of high-performance liquid chromatography (HPLC) molecular exclusion (Protein Pack 300SW column) and reverse-phase HPLC (RP-HPLC). Tricine SDS-PAGE showed that the PLA2 and crotapotins migrated as single bands with estimated molecular masses of 15 and 9 kDa, respectively. The amino acid composition of the PLA2 showed the presence of 14 half-cysteines and a high content of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA2 showed allosteric behavior, with maximal activity at pH 8.3 and 35-40 degrees C. C. d. cascavella PLA2 required Ca2+ for activity but was inhibited by Cu2+ and Zn2+ and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA2 by acting as allosteric inhibitors.
- Wang XQ, Yang J, Gui LL, Lin ZJ, Chen YC, Zhou YC
- Crystal structure of an acidic phospholipase A2 from the venom of Agkistrodon halys pallas at 2.0 A resolution.
- J Mol Biol. 1996; 255: 669-76
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The crystal structure of acidic phospholipase A2 from the venom of Agkistrodon halys pallas has been determined by molecular replacement at 2.0 A resolution to a crystallographic R-factor of 0.157. The overall structure of the molecule is very similar to those of other phospholipase A2 species of known structure. The catalytic site, the hydrophobic channel and the N-terminal region show greatest structural conservation. The Ca(2+)-binding region has a conformation that resembles closely that of bovine PLA2 rather than Crotalus atrox PLA2. Compared with other PLA2 species, the conformation of the C-terminal ridge shows significant difference due to the insertion of two residues. A unique aromatic patch appears on one face of the molecules, surrounded by two acidic residues, the relevant features of this structure and their possible biological implications are discussed.
- Suzuki A, Matsueda E, Yamane T, Ashida T, Kihara H, Ohno M
- Crystal structure analysis of phospholipase A2 from trimeresurus flavoviridis (Habu snake) venom at 1.5 A resolution.
- J Biochem (Tokyo). 1995; 117: 730-40
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The crystal structure of dimeric phospholipase A2 (PLA2) from the venom of Habu snake, Trimeresurus flavoviridis, has been determined by the molecular replacement method, and has been refined at 1.5 A resolution to an R-factor of 0.175. In the crystal, T. flavoviridis PLA2 forms a dimer using two 14 kDa subunits related by a pseudo 2-fold axis. Along the axis, the dimer has a narrow channel passing through it. Although no calcium ion is present in the calcium binding site, the peptide-chain folding of the subunits, the conformation of the catalytic residues, and the hydrogen-bonding network around the active sites are almost identical to those of the group I/II monomeric or dimeric PLA2s. The catalytic residues in both subunits are buried in the interior of the dimer and are inaccessible to substrate from the bulk solvent. In addition, the subunits of the dimer interact with each other at the hydrophobic region of the molecular surface where the entrance to the active site opens and where PLA2 is presumed to interact with the phospholipid of the substrate. Therefore, it is inferred that dimerization of T. flavoviridis PLA2 is the result of free-energy minimization by excluding the hydrophobic molecular surface from the aqueous solvent, rather than being required for the enzymatic function.
- Machado OL, Oliveira-Carvalho AL, Zingali RB, Carlini CR
- Purification, physicochemical characterization and N-terminal-amino acid sequence of a phospholipase A2 from Bothrops jararaca venom.
- Braz J Med Biol Res. 1993; 26: 163-6
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Snake venoms usually contain multiple molecular forms of phospholipase A2 enzymes (phosphatide acyl hydrolase, E.C. 3.1.1.4; PLA2). Phospholipases A2 induce a wide range of pharmacological effects which may depend or not on the hydrolysis of phospholipids. In this study, a PLA2 from Bothrops jararaca venom was purified to homogeneity by gel filtration on a Sephacryl S-200 column, followed by FPLC reverse-phase chromatography on a Pep-RPC HR 5/5 column (yield 1.63% of venom protein). The PLA2 activity of the fractions was determined by indirect hemolysis using hen's egg yolk lecithin as substrate. The enzyme is an acidic protein with PI 4.5 and an apparent molecular weight of 14,200, as estimated by gel filtration on a Superose 12 FPLC column. Similar properties have been described for PLA2 from other snake venoms. The N-terminal-sequence of the purified protein was NLMQFETMIMXXAGQ. These partial sequence data show a high degree of homology between the B. jararaca PLA2 and the enzymes from other snake venoms as well as bovine pancreatic PLA2.
- Kumar VB
- Cloning and expression of rabbit pancreatic phospholipase A2.
- Biochem Biophys Res Commun. 1993; 192: 683-92
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Pancreatic phospholipase A2 (PLA2; E.C. 3.1.1.4) has been cloned from a gt11 library made from poly A+ RNA of adult rabbit pancreas. PLA2 catalyzes the hydrolysis of the 2-acyl ester bond of 3-sn-phosphoglycerides. As the rabbits are classically used for the study of diet induced changes in lipid metabolism, as a prelude to studying the diet and age dependent changes in this enzyme, we have undertaken to clone it from a rabbit pancreatic library. Three full length clones were obtained from the rabbit pancreatic library when probed with a synthetic oligonucleotide derived from the conserved portion of the molecule. One of these clones is completely sequenced and analyzed. The sequence consists of 606 nucleotides with an open reading frame of 441 nucleotides, coding for 147 amino acids which include a 15 amino acid leader peptide and a seven amino acid propeptide. Northern blot analysis revealed a major mRNA band at 600bp. When compared to phospholipases A2 of other species, rabbit PLA2 exhibited considerable conservation both at nucleotide and the protein level. In vitro translation of synthetic mRNA obtained from T7 polymerase transcription of the cDNA yielded a protein of apparent molecular weight of 15kd similar to that predicted from its primary structure.
- Welches W, Reardon I, Heinrikson RL
- An examination of structural interactions presumed to be of importance in the stabilization of phospholipase A2 dimers based upon comparative protein sequence analysis of a monomeric and dimeric enzyme from the venom of Agkistrodon p. piscivorus.
- J Protein Chem. 1993; 12: 187-93
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Phospholipases A2 may exist in solution both as monomers and dimers, but enzymes that form strong dimers (KD approximately 10(-9) M) have been found, thus far, only in venoms of the snake family Crotilidae. The complete amino acid sequences of a basic monomeric and an acidic dimeric phospholipase A2 from Agkistrodon piscivorus piscivorus (American cottonmouth water moccasin) venom have been determined by protein sequencing methods as part of a search for aspects of structure contributing to formation of stable dimers. Both the monomeric and dimeric phospholipases A2 are highly homologous to the dimeric phospholipases A2 from Crotalus atrox and Crotalus adamanteus venoms, and both have the seven residue carboxy-terminal extension characteristic of the crotalid and viperid enzymes. Thus, it is clear that the extension is not a prerequisite for dimerization. Studies to date have revealed two characteristic features of phospholipases A2 that exist in solution as strong dimers. One is the presence in the dimers of a Pro-Pro sequence at position 112 and 113 which just precedes the seven residue carboxy-terminal extension (residues 116-122). The other is a low isoelectric point; only the acidic phospholipases A2 have been observed, thus far, to form stable dimers. These, alone or together, may be necessary, though not sufficient conditions for phospholipase A2 dimer formation. Ideas regarding subunit interactions based upon crystallographic data are evaluated relative to the new sequence information on the monomeric and dimeric phospholipases A2 from A.p. piscivorus venom.
- Fremont DH, Anderson DH, Wilson IA, Dennis EA, Xuong NH
- Crystal structure of phospholipase A2 from Indian cobra reveals a trimeric association.
- Proc Natl Acad Sci U S A. 1993; 90: 342-6
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Phospholipase A2 (PLA2) from Indian cobra venom (Naja naja naja) was crystallized from ethanol in space group P4(3)2(1)2 in the presence of Ca2+. The x-ray crystal structure was determined to 2.3-A resolution by molecular replacement techniques using a theoretical model constructed from homologous segments of the bovine pancreatic, porcine pancreatic, and rattlesnake venom crystal structures. The structure was refined to an R value of 0.174 for 17,542 reflections between 6.0- and 2.3-A resolution (F > 2 sigma), including 148 water molecules. The 119-amino acid enzyme has an overall architecture strikingly similar to the other known PLA2 structures with regions implicated in catalysis showing the greatest structural conservation. Unexpectedly, three monomers were found to occupy the asymmetric unit and are oriented with their catalytic sites facing the pseudo-threefold axis with approximately 15% of the solvent accessible surface of each monomer buried in trimer contacts. The majority of the interactions at the subunit interfaces are made by residues unique to PLA2 sequences from cobra and krait venoms. The possible relevance of this unique trimeric structure is considered.
- Christensen IT, Jorgensen FS, Svensson LA, Hogberg T
- Three-dimensional structures of human phospholipase A2 from pancreas and synovial fluid by model building.
- Drug Des Discov. 1993; 10: 101-13
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Three-dimensional structures of the enzyme phospholipase A2 (PLA2) from human pancreas and from human synovial fluid were constructed by model building based on high-resolution X-ray crystallographic structures and homology considerations. The structure of the human pancreatic PLA2 was based on the X-ray structure of the highly homologous bovine pancreatic PLA2 (Type I) by amino acid substitution and modification of the C-terminal part followed by geometry relaxation. The structure of the PLA2 from human synovial fluid was constructed from the X-ray structure of PLA2 from Crotalus atrox (Type II) by modification of the calcium binding loop, amino acid substitution, and insertion of two additional amino acid residues followed by geometry relaxation. The structure of the two human PLA2's have been compared and their use as targets for rational design of enzyme inhibitors discussed.
- Tomoo K et al.
- Interaction mode of n-dodecylphosphorylcholine, a substrate analogue, with bovine pancreas phospholipase A2 as determined by X-ray crystal analysis.
- Biochem Biophys Res Commun. 1992; 187: 821-7
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Three-dimensional structure of a bovine pancreas phospholipase A2 (PLA2) crystal complexed with n-dodecylphosphorylcholine (n-C12PC), a substrate-type inhibitor, has been determined by the X-ray diffraction method. The present conventional R value is 0.275 at 2.3A resolution. The binding mode of n-C12PC to the PLA2 was clearly indicated, where the dodecyl chain was stably held by the hydrophobic contacts with the N-terminal region of PLA2 (Leu-2, Phe-5, and Ile-9), and the choline moiety was contacted with the hydrophobic space created by the side chains of Lys-53 and 56. The present result indicates that remarkable changes from the native PLA2 structure are caused at the N-terminal and middle (residues 60 to 70) regions by the binding of n-C12PC to the enzyme.
- Siddiqi AR, Zaidi ZH, Jornvall H
- Purification and characterization of two highly different group II phospholipase A2 isozymes from a single viperid (Eristocophis macmahoni) venom.
- Eur J Biochem. 1991; 201: 675-9
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Two phospholipase A2 isozymes have been purified from leaf-nosed viper by gel permeation chromatography followed by reverse-phase HPLC and cation-exchange FPLC. Both enzymes contain seven pairs of half-cystine, typical of group II phospholipase A2. Surprisingly large differences, affecting both N- and C-terminal regions, exist between the two isozymes purified from the same snake venom. Exchanges occur at no less than 27 of 121 positions (22%), suggesting the possible existence of two genes for phospholipase A2. The residue identity with the enzymes from other Viperidae species is also low, only 44-48%, indicating extensive variations of this protein structure at large. Functionally, the present isozymes do not possess the cationic regions ascribed to myotoxicity and anti-coagulant effects of the enzyme.
- Vandermeers A, Vandermeers-Piret MC, Vigneron L, Rathe J, Stievenart M, Christophe J
- Differences in primary structure among five phospholipases A2 from Heloderma suspectum.
- Eur J Biochem. 1991; 196: 537-44
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Five increasingly anionic phospholipases A2 (Pa1-Pa5) exist in the venom of the lizard Heloderma suspectum. We recently elucidated the sequence of Pa5, the most abundant and most active variant, towards emulsified phosphatidylcholines. Here we present the primary structures of Pa2, Pa3 (subvariants a and b) and Pa4, based on Edman degradation of tryptic, endoproteinase Arg-C and chymotryptic fragments of the reduced and S-carboxymethylated proteins. Pa1-Pa5, considered collectively, belong to an original class of secretory phospholipases A2 with 141-143 residues, a short hydrophobic N-terminus, 10 half-cystine residues and an extended C-terminus. The only known phospholipase A2 with characteristics close enough to be a member of the same class is that present in the venom from the insect Apis mellifera. More specifically, the sequences of Pa3 and Pa5 are almost identical, and those of Pa2 and Pa4 are also quite similar. Both groups diverge enough to indicate the translation of two mRNA species in the venom gland. The primary structure of Pa3 reveals the existence of subvariants a and b, the sequence of which is identical to that previously defined for Pa5, except that the C-terminal tripeptide GEG in Pa5 is replaced by the dipeptide GE in Pa3a and the tetrapeptide GEGR in Pa3b, Pa4, when compared to Pa5, shows 21 substitutions with a cluster of five modified amino acids in positions 40-44, immediately after the catalytic segment amino acids 30-39, and added changes scattered before the C-terminus. Pa2 differs from Pa4 only by the absence of the Gly142 C-terminal residue. The 15% difference in primary structure observed between the Pa3-Pa5 and Pa2-Pa4 subgroups might be largely responsible for their distinct biological properties.
- Holland DR et al.
- The crystal structure of a lysine 49 phospholipase A2 from the venom of the cottonmouth snake at 2.0-A resolution.
- J Biol Chem. 1990; 265: 17649-56
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The crystal structure of a lysine 49 variant phospholipase A2 (K49 PLA2) has been determined at 2.0-A resolution. This particular phospholipase A2, purified from the venom of the eastern cottonmouth (Agkistrodon piscivorus piscivorus), a North American pit viper, differs significantly from others studied crystallographically because of replacement of the aspartate residue at position 49, whose side chain is important in calcium binding, by lysine. The crystallographic analysis of K49 PLA2 was undertaken to assess the structural ramifications of this substitution, particularly as they affect the binding mechanism of both the calcium cofactor and the phospholipid substrate. The protein crystals are tetragonal, space group P4(1)2(1)2, with unit cell dimensions of a = b = 71.7 (1) and c = 57.8 (3) A. Preliminary phases were obtained by molecular replacement techniques with a search model derived from the refined 2.5-A structure of a rattle-snake venom phospholipase A2 (Brunie, S., Bolin, J., Gewirth, D., and Sigler, P. B. (1985) J. Biol. Chem. 260, 9742-9749). The starting model gave an initial crystallographic RF of 0.526 (RF = sigma parallel to Fo /-/ Fc parallel to /sigma/Fo/). The structure was refined against all data to 2.0-A resolution. The final RF is 0.158. The final model includes 150 discrete water molecules. The K49 PLA2 model is composed primarily of alpha-helices joined by loops, some of which are quite extensive. Although dissimilarities are observed in the loop regions, the helical portions are very similar to those in other known phospholipase A2 structures. The proposed catalytic center (His48, Tyr73, and Asp99) is also structurally conserved. The region in K49 PLA2 corresponding to the calcium-binding site in other phospholipases A2 is occupied by the epsilon-amino group of lysine 49.
- Ventura MM
- Phospholipases A2: a dendrogram from a distance matrix based on size and hydrophobicity of the residues in their homologous sequences.
- An Acad Bras Cienc. 1990; 62: 177-81
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A distance matrix was obtained from aligned homologous sequences of 32 phospholipases A2 (EC 3.1.1.4) (24 from Elapid and 5 from Viperid venoms, and 3 from mammals), on the basis of the quantities Dij which are defined from a two-dimensional vector representation of the amino acid residues (dimensions: size and hydrophobicity). These Dij quantities were proposed in a previous paper (Ventura, M. M., (1989), An. Acad. brasil. Ci., 61: 215). A dendrogram was constructed from this distance matrix employing, for cluster analysis, the unweighted pair-group using arithmetic averages. Two groups of phospholipases A2: a) Elapid venom enzymes together with the three mammalian pancreatic enzymes (bovine, equine and porcine), and b) Viper venom enzymes (Crotalus, Trimeresurus and Bitis enzymes) can be well distinguished in the topology of the dendrogram. The Elapid group of enzymes is divided into two subgroups: a) Naja, Hemachatus and Bungarus venom enzymes, and b) Notechis, Laticauda, Enhydrina and Oxyuranus venom enzymes. It is observed that there is a close similarity between the mammalian pancreatic phospholipases A2 and the enzymes from Naja, Hemachatus and Bungarus. These results are similar to those reported by Dufton and Hider (Eur. J. Biochem., 137:545 (1983] which were obtained from the distance matrix based on minimum mutation distance between 25 selected residue positions in the pairwise compared sequences.
- Demaret JP, Chwetzoff S, Brunie S
- Dimeric character of a basic phospholipase A2 from cobra venom: experimental and modelling study.
- Protein Eng. 1990; 4: 171-6
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When it is gel filtered on Sephadex in the absence of calcium ions, basic phospholipase A2 from Naja nigricollis venom elutes as a dimer. In order to study the possibility of this dimerization from a structural point of view, three-dimensional models of both monomeric and dimeric N. nigricollis phospholipases A2 have been graphically built on the basis of homologies with the phospholipases A2 from pancreatic bovine and Crotalus atrox venom. The building of a dimeric model is made possible by the deletion of a particular loop of the bovine structure. The predicted models of N. nigricollis phospholipase A2 have been checked using molecular mechanics and molecular dynamics techniques according to a suitable protocol which has been developed starting from refined X-ray structures of phospholipases A2 as the test case. The observed stability of the dimeric model, in the absence of calcium, agrees with the hypothesis of the dimerization of the basic phospholipase A2. Particularly, Arg31, which replaces the hydrophobic residue present in pancreatic bovine and C.atrox venom phospholipases A2, contributes to this stability.
- Shafqat J, Beg OU, Jornvall H, Zaidi ZH
- Phospholipase A2 from cobra (Naja naja naja) venom. Primary structure and subspecies variation.
- Protein Seq Data Anal. 1989; 2: 451-2
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The primary structure of phospholipase A2 of the major race of Indian cobra has been determined. Together with previous data on other subforms, it establishes subspecies variations at no less than 20 of the 119 positions in the protein. These variations are large, not only in number but in several cases also regarding properties of the residues involved. Nevertheless, all structures are compatible with largely unaltered enzyme properties.
- Seilhamer JJ et al.
- Novel gene exon homologous to pancreatic phospholipase A2: sequence and chromosomal mapping of both human genes.
- J Cell Biochem. 1989; 39: 327-37
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We described previously the cloning and DNA sequence of the human gene encoding pancreatic phospholipase A2 [DNA 5, 519]. When pancreatic phospholipase A2 (PLA2) cDNA was used to screen a human genomic library, two classes of clones were obtained. One class encoded the pancreatic enzyme, and a second class encoded one exon of an apparently related PLA2. No additional PLA2 gene exons displayed sufficient homology to be detected by the probe. A homologous sequence in both rat and porcine genomic DNA was detected by DNA blot hybridization, and the corresponding gene fragments were cloned and sequenced. Within the deduced amino acid sequences, the presence of known functional residues along with the high degree of interspecies conservation suggests the genes encode a functional PLA2 enzyme form. The encoded sequence lacks Cys11, as do the "type II" viperid venom and other nonpancreatic mammalian PLA2 enzymes. The sequence is distinct from porcine intestinal PLA2 and appears not to be a direct homolog of the recently published rabbit ascites and rat platelet enzymes. Hybridization of DNA probes containing sequences from these genes to genomic DNA blots of mouse/human somatic cell hybrids permitted chromosomal assignment for both. The pancreatic gene mapped to human chromosome 12, and the homologous gene mapped to chromosome 1.
- Noel JP, Tsai MD
- Phospholipase A2 engineering: design, synthesis, and expression of a gene for bovine (pro)phospholipase A2.
- J Cell Biochem. 1989; 40: 309-20
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A gene coding for the (pro)phospholipase A2 (PLA2) from bovine pancreas has been designed, synthesized, and expressed in Escherichia coli. The gene was designed with a variety of restriction sites that will facilitate future mutagenesis studies. Codons occurring frequently in prokaryotic systems were chosen whenever possible. The total gene spans 404 base pairs and was divided into 33 oligonucleotides. The gene was constructed in two halves of 224 and 180 base pairs from the oligonucleotides by the shotgun ligation technique using pBSM13- as the cloning vehicle. The two fragments were then ligated and cloned into pBSM13- to complete the gene. The (pro)PLA2 gene was then verified by restriction site mapping and dideoxy sequencing. The gene was expressed to high levels from a high copy number vector, designated as pJPN, derived from the E. coli secretion vector pIN-III-ompA3. Although the protein failed to be excreted and was in the form of insoluble inclusion body, active PLA2 could be obtained by renaturation of the inclusion body pellet followed by tryptic activation, which removes the signal sequence and the pro-peptide of proPLA2. The PLA2 thus obtained reacted with the antisera raised against the natural PLA2 purified from bovine pancreas, and the specific activity of the expressed PLA2 was identical to that of the natural PLA2. The shotgun ligation and synthetic gene approaches are simple and inexpensive and can be adapted to express most of the enzymes in the phospholipase A2 family.
- Kuipers OP et al.
- Enhanced activity and altered specificity of phospholipase A2 by deletion of a surface loop.
- Science. 1989; 244: 82-5
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Protein engineering and x-ray crystallography have been used to study the role of a surface loop that is present in pancreatic phospholipases but is absent in snake venom phospholipases. Removal of residues 62 to 66 from porcine pancreatic phospholipase A2 does not change the binding constant for micelles significantly, but it improves catalytic activity up to 16 times on micellar (zwitterionic) lecithin substrates. In contrast, the decrease in activity on negatively charged substrates is greater than fourfold. A crystallographic study of the mutant enzyme shows that the region of the deletion has a well-defined structure that differs from the structure of the wild-type enzyme. No structural changes in the active site of the enzyme were detected.
- Kuchler K, Gmachl M, Sippl MJ, Kreil G
- Analysis of the cDNA for phospholipase A2 from honeybee venom glands. The deduced amino acid sequence reveals homology to the corresponding vertebrate enzymes.
- Eur J Biochem. 1989; 184: 249-54
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A cDNA expression library was constructed from worker bee venom glands and screened with an antibody against phospho lipase A2. The nucleotide sequence of a positive clone with the largest insert showed an open reading frame that codes for part of the signal peptide, the pro-region and the entire mature enzyme of the bee venom phospholipase A2 precursor. This sequence differs in the central region from the one determined by Shipolini et al. [FEBS Lett. 17, 39-40 (1971)] in showing, among other exchanges, two additional cysteines. The revised sequence of bee venom phospholipase is similar to the pancreatic enzyme in the spacing of cysteines and the presence of several amino acids known to be part of the active site or the Ca2+-binding region in identical positions. Moreover, these parts of the bee protein can be fitted into the three-dimensional structure determined for the bovine pancreatic phospholipase A2 [Dijkstra et al. (1981) Nature 289, 604-606]. Contrary to earlier suggestions, we therefore conclude that the bee venom enzyme shows some homology to phospholipases from mammalian pancreas and snake venoms.
- Tojo H, Ono T, Kuramitsu S, Kagamiyama H, Okamoto M
- A phospholipase A2 in the supernatant fraction of rat spleen. Its similarity to rat pancreatic phospholipase A2.
- J Biol Chem. 1988; 263: 5724-31
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Rat spleen supernatant contained two forms of calcium-dependent cellular phospholipase A2 which could be separated from each other by TEAE-cellulose chromatography. The phospholipase A2, named PLA2 S-1, present in the major flow-through fraction was purified to homogeneity. The structural and catalytic properties of splenic PLA2 S-1 were systematically compared with those of rat pancreatic phospholipase A2. Structural evidence, including the sequence of the N-terminal 32 residues, peptide maps obtained on Achromobacter protease I digestion and cyanogen bromide cleavage, and the amino acid composition, showed the close similarity of the two enzymes. Their catalytic and immunochemical properties were also similar. These results demonstrated the existence of a pancreatic type phospholipase A2 in a non-pancreatic organ as a member of the cellular phospholipases A2 and suggest the potential functional involvement of pancreatic type phospholipase A2 in cellular phospholipid metabolism.
- Tojo H, Ono T, Okamoto M
- A pancreatic-type phospholipase A2 in rat gastric mucosa.
- Biochem Biophys Res Commun. 1988; 151: 1188-93
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A phospholipase A2, which is immuno-crossreactive with the anti-rat pancreatic phospholipase A2 antibody, is present in rat gastric mucosa. The content of the enzyme in the gastric mucosa was comparable to that in the pancreas, but the specific activity in the gastric mucosa homogenate (60.7 +/- 19.5 nmol/min/mg) was higher than that in the pancreas homogenate (3.16 +/- 0.77 nmol/min/mg). A greater proportion of the enzyme was found in the particulate fraction. The gastric enzyme and its proenzyme were purified from the supernatant. The amino acid sequence of the N-terminal 15 residues of the gastric enzyme was determined and found to be identical with that of rat pancreatic phospholipase A2. Like the pancreatic proenzyme, the gastric proenzyme was activated on trypsin treatment.
- Hayakawa M, Kudo I, Tomita M, Nojima S, Inoue K
- The primary structure of rat platelet phospholipase A2.
- J Biochem (Tokyo). 1988; 104: 767-72
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In our previous report (Hayakawa, M., Kudo, I., Tomita, M., & Inoue, K. (1988) J. Biochem. 103, 263-266), we have shown that phospholipases A2 purified from rat platelet membrane fractions and an extracellular medium of thrombin-stimulated rat platelets were essentially identical to each other. Both purified enzymes were digested with proteases, and the resulting peptides were subjected to primary sequence determination. The sequence analysis of the HPLC-separated peptides and the alignment of the sequences showed a tentative primary structure of rat platelet phospholipase A2, which was composed of 125 amino acid residues. It showed 47% homology with snake venom Agkistrodon halys blomhoffii phospholipase A2.
- Thuren T, Tulkki AP, Virtanen JA, Kinnunen PK
- Triggering of the activity of phospholipase A2 by an electric field.
- Biochemistry. 1987; 26: 4907-10
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In this paper we show that the action of phospholipase A2 can be triggered by applying an electric field across a 1,2-didodecanoyl-sn-3-phosphoglycerol monolayer residing between an alkylated silicon surface and water. When the silicon wafer served as a cathode, rapid activation of porcine pancreatic phospholipase was observed and did depend on the magnitude of the applied potential. The degree of activation was different for the pancreatic phospholipase A2 and snake and bee venom enzymes. Maximally, a 7-fold activation of pancreatic phospholipase A2 was observed when the applied potential was 75 V. The effective field over the lipid film could be estimated to be approximately 25-175 mV, i.e., in the range of membrane potentials found in cells. On the basis of these results, we suggest that changes in membrane potential might be an important factor in the regulation of the action of intracellular phospholipases A2 in vivo.
- Mancheva I, Kleinschmidt T, Aleksiev B, Braunitzer G
- Sequence homology between phospholipase and its inhibitor in snake venom. The primary structure of phospholipase A2 of vipoxin from the venom of the Bulgarian viper (Vipera ammodytes ammodytes, Serpentes).
- Biol Chem Hoppe Seyler. 1987; 368: 343-52
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The amino-acid sequence of phospholipase A2 from the neurotoxin vipoxin of the Bulgarian Viper (Vipera ammodytes ammodytes, Serpentes) is presented. The enzyme consists of 122 amino-acid residues including 7 disulfide bonds and thus belongs to phospholipases A2 group IIA. The sequence was determined by automatic Edman degradation of the intact chain and of the peptides obtained after tryptic hydrolysis of the oxidized chain. The short cleavage time of 30 min and another limited tryptic digestion of the oxidized and citraconylated chain provided overlapping peptides. Sequencing was done with liquid- and gas-phase sequenators. The complete alignment of all peptides was facilitated by the high degree of homology with known viperid venom phospholipases A2. In common with mammalian phospholipases, the tryptophan residue in position 30 (essential for enzymatic activity) as well as the histidine in position 47 in the active site are present. Vipoxin phospholipase A2 shows 53.3% homology with another phospholipase A2 from Vipera ammodytes ammodytes venom (Ammodytoxin B), whereas 62% homology was found between both subunits of vipoxin phospholipase A2 and its inhibitor. This high degree of identity can be accounted for in terms of a common origin by gene duplication.
- Tsai IH, Liu HC, Chang T
- Toxicity domain in presynaptically toxic phospholipase A2 of snake venom.
- Biochim Biophys Acta. 1987; 916: 94-9
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About 42 complete amino-acid sequences of phospholipases A2 (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) are known, including those of 13 presynaptically toxic enzymes, but the structural features responsible for the neurotoxicity and distinguishing the toxins from the non-neurotoxic enzymes are far from being clear. In this study, we examined the charged-residue distributions and hydrophobic characteristics based on the sequence data and the predicted tertiary structure and proposed a possible toxicity domain. We found that the presynaptically toxic enzymes have three or four more basic amino-acid residues than the non-neurotoxic enzymes at positions 59, 60, 65, 70-73 and 97 or 98. These residues appear to cluster near the surface region at the N-terminal side. The cationic nature of this basic cluster in the toxin is enhanced by the alpha-amino group of the N-terminus and the dipole moment of helices 96-110 and 1-10. Moreover, these toxic-site residues are usually associated with hydrophobic regions at 1-7, 64-81 and 97-109.
- Hayakawa M, Horigome K, Kudo I, Tomita M, Nojima S, Inoue K
- Amino acid composition and NH2-terminal amino acid sequence of rat platelet secretory phospholipase A2.
- J Biochem (Tokyo). 1987; 101: 1311-4
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The amino acid composition and partial NH2-terminal amino acid sequence of the phospholipase A2 secreted by stimulated rat platelets were determined. The most predominant amino acid in the phospholipase A2 was cysteine followed by lysine, suggesting that it is a basic one. This finding is consistent with its high affinity to a cation exchange column. The NH2-terminal 24 amino acids were found to be as follows: X-Leu-Leu-Glu-Phe-Gly-Gln-Met-Ile-Leu-Phe-Lys-Thr-Gly-Lys-Arg-Ala-Asp- Val-Ser-Tyr-Gly-Phe-Tyr-Gly- The enzymes contains 5Phe, 8Met, 9Ile, 24Tyr, and 25Gly residues, all of which are conserved in the sequenced pancreatic phospholipase A2. This is the first report of the tentative characterization of a eukaryotic phospholipase A2, the cellular source of which is known, i.e., it does not originate from a venom or the pancreas.
- Ritonja A, Gubensek F
- Ammodytoxin A, a highly lethal phospholipase A2 from Vipera ammodytes ammodytes venom.
- Biochim Biophys Acta. 1985; 828: 306-12
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The amino acid sequence of ammodytoxin A, the most toxic presynaptically active phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined. The primary structure was deduced from peptides obtained by Staphylococcus aureus proteinase and trypsin digestion of reduced and carboxymethylated protein and from the automated Edman degradation of the N-terminal part of the non-reduced molecule. According to the sequence, the enzyme classifies to the subgroup IIA of the phospholipase A2 family of enzymes. The location of basic residues believed to be responsible for the toxic activity of presynaptically active phospholipases differs substantially from those in the highly toxic enzymes of other subgroups. Comparison of the sequence with sequences of other snake venom enzymes indicates that the toxic site(s) may not be the same in all subgroups of presynaptically active phospholipases.
- Okamoto M, Ono T, Tojo H, Yamano T
- Immunochemical relatedness between secretory phospholipase A2 and intracellular phospholipase A2.
- Biochem Biophys Res Commun. 1985; 128: 788-94
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The immunochemical relationship between rat pancreatic phospholipase A2 and rat splenic phospholipase A2 was examined with the use of anti-rat pancreatic phospholipase A2 antibody as a probe. The immunoelectrophoretic patterns showed that the antibody cross-reacted with the splenic enzyme. The immuno-crossreactivity was also shown by counter immunoelectrophoresis. The splenic phospholipase A2, whether it was purified from the cytosolic fraction or the microsomal fraction, formed an immunoprecipitin band with the anti-pancreatic phospholipase A2 antibody. The antibody was shown to inhibit the activity of the pancreatic phospholipase A2 as well as that of the splenic phospholipase A2.
- Wurl M, Kunze H
- Purification and properties of phospholipase A2 from human seminal plasma.
- Biochim Biophys Acta. 1985; 834: 411-8
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The soluble Ca2+-dependent phospholipase A2 (EC 3.1.1.4) was purified 6500-fold with a yield of about 20% from human seminal plasma. The successive purification steps comprised gel filtration, affinity chromatographies and micropartition. The final preparation consisted of two proteins in about equal quantities with molecular weights of 12000 and 14000, according to SDS-polyacrylamide slab gel electrophoresis. As yet these two proteins can not be separated without complete loss of activity. Apparent kinetic parameters have been determined for the purified preparation with different substrates (Vmax = 494 U/mg, and Km = 1.25 X 10(-4) M long-chain phosphatidylethanolamine; Vmax = 7.4 U/mg, and Km = 2.5 X 10(-5) M long-chain phosphatidylcholine; Vmax = 7196 U/mg and Km = 8.32 X 10(-4) M dioctanoylphosphatidylcholine). The enzymatic activity was not affected by diisopropylfluorophosphate and thiol reagents but it was inhibited by higher concentrations of nonionic and ionic (except taurocholate) detergents and by the alkylating reagent p-bromophenacyl bromide. Although the seminal enzyme functionally strongly resembles the pancreatic phospholipase A2, no immunochemical relationship was observed; anti-pancreatic phospholipase A2 IgGs did not inhibit seminal phospholipase A2. Similarly, partially purified phospholipase A2 from horse seminal fluid was not affected by antibodies raised against horse pancreatic phospholipase A2.
- Renetseder R, Brunie S, Dijkstra BW, Drenth J, Sigler PB
- A comparison of the crystal structures of phospholipase A2 from bovine pancreas and Crotalus atrox venom.
- J Biol Chem. 1985; 260: 11627-34
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The refined high resolution crystal structure of the bovine phospholipase A2 was compared with its counterpart from the venom of Crotalus atrox, the western diamondbacked rattlesnake. The strong similarity in their backbone conformations forms the basis of a common numbering system for the amino acid sequence. The three common major helices and much of the extended chain form a nearly identical "homologous core" structure. The variations in conformation usually arise from deletions/insertions or en bloc shifts of structural units. The exception to this is part of the highly conserved calcium-binding loop; however, this is to be expected as 1) there is no calcium ion sequestered in the venom dimer as there is in the case of the bovine enzyme and 2) two side chains in that segment form dimer-stabilizing interactions between the subunits of the C. atrox enzyme. The absolutely conserved catalytic network of hydrogen-bonded side chains formed by His 48, Tyr 52, Tyr 73, and Asp 99, as well as the hydrophobic wall that shields it, are virtually superimposable in the two structures. However, the details of the structural relationship between the amino terminus and the catalytic network differ in the two species and the ordered water molecules thought to be either functionally or structurally important in the pancreatic enzymes are not found in the crystal structure of the phospholipase A2 from C. atrox. The most striking difference from a functional standpoint is the fact that the surface depression in the region of the catalytic network that has been commonly considered the active site is shielded substantially in forming the intersubunit contact surface of the dimeric venom enzyme.
- Dijkstra BW, Kalk KH, Drenth J, de Haas GH, Egmond MR, Slotboom AJ
- Role of the N-terminus in the interaction of pancreatic phospholipase A2 with aggregated substrates. Properties and crystal structure of transaminated phospholipase A2.
- Biochemistry. 1984; 23: 2759-66
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A free N-terminal alpha-NH3+ group is absolutely required for full catalytic activity of phospholipase A2 on aggregated substrates. To elucidate how this alpha-NH3+ group triggers catalytic activity, we specifically transaminated this group in various pancreatic phospholipases A2. Porcine, porcine iso-, equine, human, ovine, and bovine phospholipases A2 all loose catalytic activity on micellar substrates due to the inability of the transaminated proteins to bind to neutral micellar substrate analogues, as was found for the zymogens. Loss of activity is pseudo first order, the rate constants being different for the enzymes studied. The transaminated phospholipases A2 have an intact active site, as catalytic activities on monomeric substrates are comparable to those of the respective zymogens. The X-ray structure of transaminated bovine phospholipase A2 at 2.1-A resolution shows that the N-terminal region and the sequence 63-72 in this protein are more flexible than in the native enzyme. Also, in this respect, the transaminated enzyme very much resembles the zymogen structure. In good agreement with this, it was found by photochemically induced dynamic nuclear polarization 1H NMR that aromatic resonances of Trp-3 and Tyr-69 are affected by transamination. In addition, fluorescence spectroscopy of the unique Trp-3 in transaminated bovine phospholipase A2 revealed a red shift of the emission maximum indicative of a more polar environment of Trp-3 in the transaminated phospholipase A2 as compared to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
- Tuichibaev MU, Iakubov IT, Rakhimov MM, Tashmukhamedov BA
- [Properties of phospholipase A2 from the venom of the large hornet Vespa orientalis]
- Biokhimiia. 1984; 49: 1546-55
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Some properties (catalytic and hemolytic activity, pH and temperature optima, stability, substrate specificity, effects of detergents and metal ions, N-terminal sequence, chemical modification of histidine in the enzyme active center, etc.) of phospholipase A2 from hornet (Vespa orientalis) venom were studied. It was shown that phospholipase A2 from hornet venom differs essentially from other enzymes of this species in terms of stability, catalytic properties and structural features. The active center of the enzyme contains an essential histidine residue, similar to other phospholipases A2 from various sources. Unlike other known forms of phospholipase A2, the enzyme under study exerts a pronounced hemolytic action. The hemolysis is inhibited by Ca2+ at concentrations capable of inducing the activation of the hydrolytic activity of the enzyme.
- Dehaye JP et al.
- Phospholipase A2 activity of pancreatic secretory factor, a new secretagogue isolated from the venom of Heloderma suspectum.
- FEBS Lett. 1984; 172: 284-8
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Pancreatic secretory factor (PSF), an efficient pancreatic secretagogue recently isolated from the venom of Heloderma suspectum, is shown to exert phospholipase A2 activity towards phosphatidylcholine. This activity is strictly dependent on calcium (apparent Ka 40 nM) and has an optimum pH around 9. At pH 7.4 and in the presence of calcium, PSF retains 40% of its phospholipase A2 activity. These results are compared to the calcium dependency of the secretory effect of PSF on rat pancreatic acini. Taken collectively, the present data on PSF suggest that a similar endogenous phospholipase A2 activity might be involved in the late steps of stimulus-secretion coupling in the exocrine pancreas.
- Ono T, Tojo H, Inoue K, Kagamiyama H, Yamano T, Okamoto M
- Rat pancreatic phospholipase A2: purification, characterization, and N-terminal amino acid sequence.
- J Biochem (Tokyo). 1984; 96: 785-92
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Phospholipase A2 was purified from rat pancreas by heat treatment of the homogenate and the sequential use of DEAE-Sepharose chromatography, CM-Sepharose chromatography, and reverse-phase high-performance liquid chromatography (HPLC). Prophospholipase A2 was not separated from the phospholipase A2 by CM-Sepharose chromatography under the conditions used, but it was well resolved by the reverse-phase HPLC. The enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and on analytical HPLC, and its molecular weight was estimated to be 14,000. The enzyme specifically hydrolyzed an acylester bond at the sn-2-position of the phospholipid examined. The purified enzyme has a pH optimum in the range of pH 9.5 to 10.5 and requires the presence of Ca2+ (3 mM) and sodium deoxycholate (0.1%) for optimum activity. The amino acid sequence of the first 32 residues in the N-terminal region of the enzyme was determined. The sequence revealed a marked homology with those of pancreatic phospholipases A2 of man, pig, ox, and horse, and porcine intestinal phospholipase A2 reported previously.
- Dufton MJ, Hider RC
- Classification of phospholipases A2 according to sequence. Evolutionary and pharmacological implications.
- Eur J Biochem. 1983; 137: 545-51
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The sequences of 32 phospholipases A2 (EC 3.1.1.4) were systematically compared on the basis of polypeptide chain length and similarity at selected amino acid positions around the active site. Two difference matrices were constructed and the various groupings present in the data were expressed in dendrogram form. The two methods of comparison yielded different results, and this is seen as a consequence of separate aspects of phospholipase evolution being highlighted in each case. It appears that, although Elapid snake venom phospholipases are very similar in terms of overall conformation, the area around their active sites distinguishes them into two major groups, namely the Asian Elapids and the marine/Australasian Elapids. Further, the Asian Elapids seem to have active-site vicinities which are closer to those in the mammalian pancreatic phospholipases. The relevance of the classifications to structure/activity relationships (especially beta-neurotoxicity) and phospholipase evolution is discussed.
- Dijkstra BW, Weijer WJ, Wierenga RK
- Polypeptide chains with similar amino acid sequences but a distinctly different conformation. Bovine and porcine phospholipase A2.
- FEBS Lett. 1983; 164: 25-7
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The primary structures of bovine and porcine pancreatic phospholipase A2 differ only by about 15%. Nevertheless, a 12 residue loop, with only one substitution (Val leads to Phe) has a quite different conformation, whereas the rest of the molecules have a very similar folding indeed. From this observation it is concluded that prediction of a 3-dimensional structure on the basis of sequence similarity of short segments alone might give erroneous results.
- Nishijima J, Okamoto M, Ogawa M, Kosaki G, Yamano T
- Purification and characterization of human pancreatic phospholipase A2 and development of a radioimmunoassay.
- J Biochem (Tokyo). 1983; 94: 137-47
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Human pancreatic phospholipase A2 was purified to homogeneity from pancreatic juice and a reliable radioimmunoassay for the enzyme was developed. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 14,000. Phosphatidylcholine was hydrolyzed well in an alkaline pH range, and the optimum activity was obtained at pH 9. Calcium ion was indispensable for activity. The enzyme was stable to heat treatment at 60 degrees C for 5 min. The radioimmunoassay system was highly sensitive, reproducible and specific. The dilution curves for the sera of patients with acute pancreatitis were parallel to the standard curve. In healthy individuals, serum phospholipase A2 concentrations ranged from 2.0 to 7.9 ng/ml, the average being 5.1 ng/ml (S.D.: 1.7). In patients with acute pancreatitis, significant elevations of serum phospholipase A2 contents were observed, and the highest value found was 4,000 ng/ml.
- Grataroli R, Dijkman R, Dutilh CE, van der Ouderaa F, De Haas GH, Figarella C
- Studies on prophospholipase A2 and its enzyme from human pancreatic juice. Catalytic properties and sequence of the N-terminal region.
- Eur J Biochem. 1982; 122: 111-7
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Upon tryptic activation of pure human prophospholipase A2, a heptapeptide is released from the N-terminal part of the protein yielding active phospholipase A2 (EC 3.1.1.4). Both the kinetics of the activation process and the amino acid sequence of the activation peptide strongly resemble those of pancreatic zymogens of other mammalian sources. The kinetic properties of human phospholipase A2 and its zymogen are compared with those of the corresponding porcine enzyme using substrates present at micelles, molecular dispersed solutions or as monomolecular surface films. The most obvious difference between the human and porcine phospholipase A2 is the low enzyme activity of the former protein at pH 8.0 as compared to pH 6.0, both against micellar and monomeric substrates. Neither the Ca2+ binding properties nor the inhibition of the human enzyme using haloketones can easily explain this different pH optimum. The sequence analysis of the N-terminal region of the first 40 residues is reported.
- Evstratova NG, Aianian AE, Miroshnikov AI, Serebrennikova GA, Evstigneeva RP
- [Isolation of phospholipase A2 on biospecific supports]
- Biokhimiia. 1982; 47: 1547-51
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The procedure of isolation of phospholipase A2 from Agkistrodon halys blomhoffii venom and porcine pancreas on biospecific supports of an organo-silica type with immobilized phospholipid is described. The purity of isolated enzymes was controlled by polyacrylamide gel electrophoresis, determination of the N-terminal amino acid and isoelectrofocusing. The enzyme activity was equal to 80 mumole/mg/min and 1400 mumole/mg/min for snake venom phospholipase and for pancreatic phospholipase A2, respectively.
- Dijkstra BW, Drenth J, Kalk KH
- Active site and catalytic mechanism of phospholipase A2.
- Nature. 1981; 289: 604-6
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The esterolytic enzyme phospholipase A2 specificially splits the 2-acyl linkage of phosphoglycerides in a calcium-dependent reaction. In the pancreas the enzyme occurs as a zymogen which is activated on secretion into the duodenal tract by the removal of seven amino acid residues from the N terminus by trypsin. Having refined our X-ray analysis of the crystal structure of bovine pancreatic phospholipase A2 from 2.4 A (ref. 4) to 1.7 A resolution, we now describe how the structure of the molecule may account for the specificity of the enzyme and for the sudden and dramatic change in activity when the substrate concentration passes the critical micelle concentration.
- Durand S, Clemente F, Douste-Blazy L
- A non-secretory phospholipase A2 in the rat pancreas.
- Biomedicine. 1980; 33: 19-21
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The rat pancreas shows a phospholipase activity in the absence of the tryptic activation of the zymogens. We verified that this activity was not due to the zymogen of the secretory phospholipase. Indeed unlike the prophospholipase, the enzyme responsible for the spontaneous phospholipase activity shows a higher catalytic rate at substrate concentrations higher than the critical micelle concentration. This enzyme has a positional specificity of the A2 type and possesses a molecular weight of about 9,500.
- van Scharrenburg GJ, de Haas GH, Slotboom AJ
- Regeneration of full enzymatic activity by reoxidation of reduced pancreatic phospholipase A2.
- Hoppe Seylers Z Physiol Chem. 1980; 361: 571-6
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Reduction of porcine pancreatic phospholipase A2 with 2-mercaptoethanol in the presence of 6M guanidinium hydrocholoride or 8M urea resulted in the complete disruption of all seven disulfide bridges and the concomitant loss of all enzymatic activity. Through aerobic oxidation of the reduced protein at pH 8.0 in the presence of 5 mM cysteine and 0.9M guanidinium hydrochloride up to 90% of the specific activity of the native enzyme was restored. After purification of the reoxidized phospholipase A2 by Sephadex G-75 gel filtration and chromatography on CM-cellulose pure phospholipase A2 was obtained in 80% yield. This purified reoxidized phospholipase A2 was found to be indistinguishable from the native enzyme as judged by its enzymatic activity, lipid and Ca2+ binding properties. Similar results were obtained using S-sulfonated phospholipase A2, prophospholipase A2 or Nepsilon-amidinated phospholipase A2. For the latter two proteins reoxidation was slower than that of the reduced phospholipase A2.
- Heinrikson RL, Krueger ET, Keim PS
- Amino acid sequence of phospholipase A2-alpha from the venom of Crotalus adamanteus. A new classification of phospholipases A2 based upon structural determinants.
- J Biol Chem. 1977; 252: 4913-21
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The complete amino acid sequence of Crotalus adamanteus venom phospholipase A2-alpha has been determined by analysis of the five tryptic peptides from the citraconylated, reduced, and S-[14C]carboxamidomethylated enzyme. Earlier studies (Tsao, F. H. C., Keim, P. S., and Heinrikson, R. L. (1975) Arch. Biochem. Biophys. 167, 706) provided the information necessary to align the tryptic fragments so that secondary cleavage procedures to establish overlaps were unnecessary. The subunit in the phospholipase A2-alpha dimer is a single polypeptide chain containing 122 amino acids and seven disulfide bonds. The histidine residue implicated in the active site of mammalian phospholipases is at position 47 in the C. adamanteus enzyme and is located in a domain of the molecule which is highly homologous in sequence with corresponding regions of phospholipases from a variety of venom and pancreatic sources. Comparative sequence analysis has revealed insights with regard to the function and evolution of phospholipases A2. Primary structural relationships observed among the snake venom enzymes parallel the phylogenetic classification of the venomous reptiles from which they were derived. It is proposed that phospholipases A2 of this general type be divided into two groups depending upon the presence or absence of distinctive structural features elucidated in this study.
- Slotboom AJ, de Haas GH
- Specific transformations at the N-terminal region of phospholipase A2.
- Biochemistry. 1975; 14: 5394-9
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Treatment of porcine pancreatic prophospholipase A2 with methyl acetimidate converted all lysine residues into epsilon-acetimidolysine residues. Enzymatically active epsilon-amidinated phospholipase A2 (AMPA) was obtained from the epsilon-amidinated zymogen by limited tryptic proteolysis cleaving the Arg7-Ala8 bond. AMPA was used to prepare des-Ala8-, des-(Ala8,Leu9)- and des-(ALa8),Leu9,Trp10)-AMP by successive Edman degradations, and des-(A la 8-Arg13)-AMPA by selective splitting of the Arg13-Ser14 bond by trypsin. Structural analogues of AMPA with different N-terminal amino acid residues, viz., D-Ala, beta-Ala, and Gly, have been prepared by reacting des-Ala8-AMPA with the corresponding N-t-Boc-N-hydroxysuccinimide esters of these amino acids. Similarly, the only Trp10 residue has been substituted for Phe by coupling of des-(Ala8-,Leu9,Trp10)-AMPA with N-t-Boc-L-Ala-L-Leu-L-Phe-N-hydroxysuccinimide ester. The feasibility of these substitutions has been proven unambiguously by the retroconversion of des-Ala8-AMPA and of [Ala7]AMPA into AMPA having identical enzymatic activity as the starting AMPA. The single Trp10 residue in native phospholipase A2 and its zymogen was specifically sulfenylated using 0-nitrophenyl-sulfenyl chloride. The homogenous proteins were kinetically analyzed using short-chain lecithins in the monomeric and micellar region. All modified AMPA analogues, except those in which two or more of the N-terminal amino acid residues are removed, show enzymatic activities toward monermic substrate comparable to that of AMPA, indicating that the active site region is still intact. Only [Gly8]-, [beta-Ala8]-, and [Ala8,Leu9,Phe10]AMPA exhibit a dramatic increase in enzymatic activity similar to that of AMPA upon passing the critical micellar concentration (cmc) of the substrate. From these results it can be concluded that the N-terminal region of the enzyme requires a very precise architecture in order to interact with lipid-water interfaces and consequently to display its full enzymatic activity.