Ammodytoxin A, a highly lethal phospholipase A2 from Vipera ammodytes ammodytes venom

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.     

Susceptibility of the Wolfgram Proteins and Stability of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase of Rat Brain Myelin to Limited Proteolytic Digestion

The susceptibility of proteins in the myelin membrane to proteases was studied. Lyophilized rat brain myelin suspended in water was subjected to controlled proteolytic digestion with pure trypsin (N-tosyl-L-phenylalanine chloromethyl ketone treated, 5 units/mg of myelin), and proteins remaining in the pellet were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under these conditions, large basic protein (LBP) was completely hydrolyzed in 5-10 min, proteolipid proteins remained largely intact until 60 min, whereas Wolfgram protein (WP) was progressively degraded from 10 min onward with the simultaneous appearance of a new protein band with a molecular weight of 35K. A similar pattern was obtained on treatment with chymotrypsin or subtilisin. The 35K protein band was shown to be derived from WP by its immunological cross-reactivity with WP antibodies. Western blot analysis showed that 35K protein is the only major breakdown product of WP under these conditions. Treatment with higher concentrations of trypsin (greater than 20 units/mg of myelin) resulted in the degradation of all myelin proteins. Essentially all the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) activity was observed in the myelin pellet after controlled or drastic digestion with trypsin. It is concluded that the major fragment of WP (35K) is located in the hydrophobic milieu of the bilayer, relatively inaccessible to trypsin, whereas a portion (20K) of the WP is exposed to the cytoplasmic side (major dense line), like LBP, and that peptide fragments (less than 14K) that remained in the myelin membrane lipid bilayer after trypsin digestion could exhibit CNP activity.     

EFFECT OF PROTEOLYTIC ATTACK ON THE STRUCTURE OF CNS MYELIN MEMBRANE

Rat CNS myelin particles have been incubated with trypin and acetyltrypsin under conditions which ensured a selective and substantial removal of the basic proteins leaving acidic Wolfgram and proteolipid proteins. Some trypsin became associated with the basic protein denuded pellet while no attachment of acetyltrypsin was observed. The removal of basic proteins ‘solubilized’ some myelin and produced a lighter ‘fluff’ layer on top of the myelin pellet, but this amounted to no more than 10 per cent of the total myelin lamellae. Electron microscopy indicated a more dense-straining interperiod line in a small percentage of lamellae which otherwise remained normal. Selective extraction of complex lipids with solvents of increasing polarity, nuclear magnetic resonance spectra and X-ray diffraction patterns showed no significant changes on removing basic proteins from myelin. The results are interpreted as suggesting that the basic proteins are not uniformly distributed in myelin but preferentially located in the outside layers of the myelin sheath and that they play little part in stabilizing the bulk of the myelin membrane structure.     

Activation of Brain Guanylate Cyclase by Phospholipase A2

Various pure snake venom phospholipases A2 were used for studying their effect on guanylate cyclase activity. All the phospholipases A2 tested were found to activate guanylate cyclase from a rat brain homogenate. It was shown that particulate guanylate cyclase was especially affected. Intact glial cells incubated in presence of phospholipase A2 showed also an increased guanylate cyclase activity, demonstrating that the phospholipase effect, observed in disrupted cells, occurs also at the cellular level. These results suggest that in intact cells membrane-bound phospholipase A2 activity could be involved in the modulation of the cellular cyclic GMP content.     

Amino acid sequence of a basic Agkistrodon halys blomhoffii phospholipase A2. Possible role of NH2-terminal lysines in action on phospholipids of Escherichia coli

A basic (pI = 10.2) phospholipase A2 of the venom of the snake Agkistrodon halys blomhoffii is one of a few phospholipases A2 capable of hydrolyzing the phospholipids of Escherichia coli killed by a bactericidal protein purified from human or rabbit neutrophil granules. We have shown that modification of as many as 4 mol of lysine per mole of the phospholipase A2, either by carbamylation or by reductive methylation [Forst, S., Weiss, J., & Elsbach, P. (1982) J. Biol. Chem. 257, 14055-14057], had no effect on catalytic activity toward extracted E. coli phospholipids or the phospholipids of autoclaved E. coli. In contrast, modification of 1 mol of lysine per mole of enzyme substantially reduced activity toward the phospholipids of E. coli killed by the neutrophil protein. To explore further the role of lysines in the function of this phospholipase A2, we determined the amino acid sequence of the enzyme and the incorporation of [14C]cyanate into individual lysines when, on average, 1 lysine per molecule of enzyme had been carbamylated. After incorporation of approximately 1 mol of [14C]cyanate per mole of protein, the phospholipase A2 was reduced, alkylated, and exhaustively carbamylated with unlabeled cyanate. The amino acid sequence was determined of the NH2-terminal 33 amino acids of the holoprotein and of peptides isolated after digestion with trypsin and Staphylococcus aureus V-8 protease. The protein contains 122 amino acid residues, 17 of which are lysines. The NH2-terminal region is unique among more than 30 phospholipases A2 previously sequenced because of its high content of basic residues (His-1, Arg-6, and Lys-7, -10, -11, and -15).(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid and basic protein interaction in myelin

1. Purified myelin labelled with [(3)H]myo-inositol or [1-(14)C]acetate was incubated with trypsin or acetylated trypsin at 37 degrees C, pH8.0 for 30min. 2. After incubation and centrifugation analysis of the myelin pellet showed marked digestion of basic protein on polyacrylamide-gel electrophoresis. Proteolipid and Wolfgram proteins remained unchanged. 3. A loss of 15% of total protein and loss of all classes of lipids was also found. Most significant lipid losses were phosphoinositides, phosphatidylserine and sulphatide. 4. A low-density material containing more phospholipid than cholesterol and galactolipid was isolated from the supernatant obtained after centrifugation of trypsin-treated myelin. 5. Interaction of sulphatide and myelin basic protein was shown to take place in a biphasic system. Basic protein does not form any complex either with cerebroside or cholesterol in the same solvent system. 6. The release of acidic lipids from myelin suggests that they may be linked to basic protein by ionic forces and the neutral lipids may be by lipid-lipid interactions. 7. The relevance of these studies as a model of brain degeneration is discussed.     

Sequence homology between phospholipase and its inhibitor in snake venom. The primary structure of the inhibitor of vipoxin from the venom of the Bulgarian viper (Vipera ammodytes ammodytes, Serpentes)

We are presenting the first primary structure of a snake venom inhibitor. It was isolated from the neurotoxin vipoxin of the Bulgarian Viper (Vipera ammodytes ammodytes, Serpentes) which represents a complex of a strong toxic basic protein with phospholipase A2 activity (2 isoenzymes) and the nontoxic acidic component functioning as its inhibitor. The sequence was established by automatic degradation in a liquid phase sequenator on the S-carboxymethylated chain and on the peptides obtained by tryptic hydrolysis of the oxidized chain. A limited tryptic digestion of the oxidized chain provided the necessary overlapping peptides. The inhibitor consists of 122 amino-acid residues including 14 cysteine and 10 tyrosine residues and is thus similar to the phospholipases from snake venoms. A comparison of the inhibitor sequence with the primary structure of the phospholipase A2 (CM-II) from the Horned Adder (Bitis nasicornis) venom shows a surprising homology of 52%. The identical amino acids include the cysteine and tyrosine residues and are generally accumulated in the surroundings of cysteine residues. The histidine (pos. 47) in the active center of the phospholipase A2 is substituted by glutamine in the inhibitor, but the tryptophan (pos. 30) which is essential for the enzymatic activity is present. The significant homology between enzyme and inhibitor in the vipoxin complex is believed to originate from a gene duplication. The relatively late development of the reptiles and the snake venom complex explains the highly preserved structure compared to other enzyme-inhibitor systems.     

Identification and isolation of a mammalian protein which is antigenically and functionally related to the phospholipase A2 stimulatory peptide melittin

Antibodies prepared against the phospholipase A2 stimulatory peptide melittin were used to identify and isolate a novel mammalian protein with similar functional and antigenic properties. The mammalian protein of Mr 28,000 was isolated from cell sonicates by high performance immunoaffinity chromatography and size exclusion chromatography. This stimulatory protein was stable for several months when frozen at -70 degrees C. The purified protein selectively stimulated phospholipase A2 when phosphatidylcholine was used as a substrate but had no effect on phospholipase A2 activity when phosphatidylethanolamine was used as a substrate. Furthermore, this protein had no effect on phospholipase C activity or on pancreatic or snake venom phospholipase A2. The stimulatory activity was unaffected by RNase or DNase treatment. However, boiling or trypsin digestion inactivated the phospholipase stimulatory activity. The mechanism of phospholipase A2 stimulation appeared to result from an increase in the apparent Vmax of the enzyme.     

Structural and functional properties of a phospholipase A2 purified from an inflammatory exudate

The cell-free supernatant of sterile inflammatory peritoneal exudates contains a phospholipase A2 that participates in the digestion of Escherichia coli killed by polymorphonuclear leukocytes or by the purified bactericidal/permeability increasing protein (BPI) of these cells. This phospholipase A2 has been purified, and the sequence of the NH2-terminal 39 amino acids has been determined and compared with sequences of both BPI-responsive and BPI-nonresponsive phospholipases A2 from snake venoms and mammalian pancreas. The high concentration and location of basic residues in the NH2-terminal region is a common feature of BPI-responsive phospholipases A2 and may characterize those phospholipases A2 participating in inflammatory events.     

Evidence for a phospholipid requirement in the specific binding of glucocorticoids to receptors of fibroblasts and thymic lymphocytes.

The specific steroid binding capacity of soluble preparations from mouse fibroblasts and rat thymic lymphocytes is inactivated by incubation with phospholipases. Receptor binding is drastically reduced by very low concentrations of boiled phospholipase A preparations from bee venom and snake venoms. The enzyme effect is calcium-dependent and is blocked by both phospholipid and a substrate analog that is a competitive inhibitor of phospholipase A. The specific binding capacity is also sensitive to digestion by phospholipase C. Two possible mechanisms are considered for the phospholipase A effect: (a) the receptor protein may be associated with a phospholipid component which is required for specific hormone binding; (b) phospholipase A may be producing detergent products that are indirectly inactivating the receptor. Examination of the effects of lysophosphatide on the receptor and assay of lipid phosphate in the receptor preparation do not support a mechanism based solely on detergent effects. Because phospholipase C, which does not produce detergent products, also inactivates the binding, we propose that the phospholipases may be digesting the phospholipid which is a requisite component of the glucocorticoid receptor.     

HISTOCHEMISTRY OF MYELIN XIV PERIPHERAL NERVE MYELIN PROTEINS: ELECTROPHORETIC AND HISTOCHEMICAL CORRELATIONS

Abstract— Myelin from the peripheral nervous system has been shown to contain two basic protein components and an electrophoretically slower-moving major protein, the 'J' band. The 'J' band protein cannot be selectively removed by aqueous or organic solvents and does not correspond to proteolipid or acidic protein. Histochemical stains applied to peripheral nervous systems myelin proteins separated by polyacrylamide electrophoresis indicate that 'J' band protein is analogous with the neurokeratin of the nerve sheath. Trypanophilia observed histochemically in unfixed myelin is principally due to basic proteins. With prolonged tryptic digestion 'J' band protein is degraded. Thus, previous classifications of myelin proteins based on trypsin sensitivity have been modified. All peripheral nervous system myelin proteins should be regarded as trypsin-sensitive, the basic protein being relatively more and the 'J' band protein relatively less susceptible.     

THE PROTEIN COMPOSITION OF ISOLATED MYELIN1

—Three fractions, each containing markedly different proteins, was obtained from myelin: (1) The first fraction was obtained as an insoluble residue when myelin was extracted with neutral chloroform-methanol (CM, 2:1, v/v). It was digestible with trypsin and had an amino acid composition similar to that of the acidic proteolipid protein of Wolfgkam (1966). (2) The second fraction was obtained as a precipitate by the addition of various electrolytes (KCl, NaCl, CaCl₂, MgCl₂ or HCl) to the CM (2:1 v/v) extract. This fraction consisted mainly of a basic protein which exhibited an electrophoretic mobility and amino acid composition indistinguishable from those of the basic protein obtained from white matter (Martensson and LeBaron, 1966). This procedure provided for a simple and rapid isolation of the basic protein from myelin. Depending on the conditions of precipitation, this fraction was either free of lipid or contained tri- and diphosphoinositide. The effects of different ions at differing concentrations and the yield and nature of the precipitate have been studied. (3) A third fraction remained in solution in CM (2:1, v/v) after the addition of the electrolyte. It comprised the bulk of the myelin lipids and a protein fraction which was resistant to digestion with trypsin and had an amino acid composition similar to the classical proteolipid protein of Folch-Pi and Lees (1951). The possibility of a salt-type bonding between the basic protein and the polyphosphoinositides is discussed, and values for tri- and diphosphoinositide in bovine myelin are given.     

Purification and amino acid sequence of basic protein II, a lysine-49-phospholipase A2 with low activity, from Trimeresurus flavoviridis venom

A basic protein (pI 10.3), named basic protein II, was purified to homogeneity from the venom of Trimeresurus flavoviridis (Habu snake) after four chromatographic steps. The amino acid sequence of this protein was determined by sequencing the S-pyridylethylated derivative and its peptides produced by chemical (cyanogen bromide) and enzymatic (chymotrypsin, clostripain, and Staphylococcus aureus V8 protease) cleavages. The protein consisted of 122 amino acid residues and was found to be identical in sequence to basic protein I from the same source except that Asp-58 of basic protein I is replaced by asparagine. Like basic protein I, the structural feature of basic protein II is that Tyr-28 and Asp-49 common in phospholipases A2 from snake venoms and mammalian pancreas are replaced by asparagine and lysine, respectively. Thus, basic protein II belongs to the category of lysine-49-phospholipase A2. The action of basic protein II on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine released only oleic acid, indicating that it has phospholipase A2 activity. Its molar activity toward 1,2-dilauroyl-sn-glycero-3-phosphorylcholine, however, was only 1.7% of that of T. flavoviridis phospholipase A2 isolated previously. Affinity for Ca2+ and reactivity toward p-bromophenacyl bromide of basic protein II were 8 and 5.3 times, respectively, smaller than those of phospholipase A2 from the same source, substantiating the low phospholipase A2 activity of basic protein II.     

日本蝮蛇蛇毒碱性磷脂酶A2同源物的分离及鉴定

We purified and characterizated a phospholipase A2 homologue from Agkistrodon blomhoffii ussurensis snake venom. We used Hitrap SP cation exchange and Superdex 75 columns chromatography to obtain a basic protein, used SDS-PAGE to analyse molecular mass, and IEF (Isoelectric focusing electrophoresis) IEF to identify isoelectric point. The molecular mass was 16 kDa, and the isoelectric point was 8.56. We detected its phospholipase A2 activity on egg yolk phospholipids, hemolytic activity on washed erythrocytes, and anticoagulant effect on pig platelet-rich plasma, as well as the N-terminal sequence with protein sequencer. The results showed that it had no phospholipase A2 activity and hemolytic activity, but had obvious anticoagulant effect on in witro. The N terminal sequence (21 amino acid residues) compared with other phospholipases A2 demonstrated that the protein was homogenous with BPLA2s from Agkistrodon halys Palls.     

Inactivation of phospholipase A2 and metalloproteinase from Crotalus atrox venom by direct current

To achieve our aim of understanding the interactions between direct current and enzymes in solution, we exposed reconstituted Crotalus atrox venom to direct electric current by immersing two platinum thread electrodes connected to a voltage generator (between 0 and 8 V) into a reaction mixture for a few seconds. Then, we assayed the residual activity of phospholipases A(2) (PLA(2)),metalloproteinases, and phosphodiesterases, abundant in crotaline snake venoms and relevant in the pathophysiology of envenomation, characterized by hemorrhage, pain, and tissue damage. C. atrox venom phospholipase A(2) and metalloproteinases were consistently and irreversibly inactivated by direct current (between 0 and 0.7 mA) exposure. In contrast, C. atrox venom phosphodiesterases were not affected. Total protein content and temperature of the sample remained the same. Secretory pancreatic phospholipase A(2), homologue to snake venom phospholipases A(2), was also inactivated by direct current treatment. In order to understand the structural reasoning behind PLA(2) inactivation, circular dichroism measurements were conducted on homogeneous commercial pancreatic phospholipase A(2), and it was found that the enzyme undergoes structural alterations upon direct current exposure.     

Studies on phospholipase A inhibitor in blood plasma. II. Interaction of phospholipase A inhibitor with phospholipase A and its specificity

Non-competitive inhibition of snake venom phospholipase A2 which has been exhibited by bovine plasma phospholipase A inhibitor, a kind of lipoprotein, was not observed unless the inhibitor was preincubated with the enzyme. The inhibition seemed to be due to the formation of the enzyme-inhibitor complex, which was identified by immunoelectrophoresis. The enzyme-inhibitor interaction was observed maximally on incubation at physiological pH, but not below pH 5. The inhibitor was inactivated by trypsin digestion and heat treatment. It suppressed the phospholipase A2 activities of rat blood plasma as well as of the snake venom and porcine pancreas, but not the enzyme activities such as those of phospholipase C of Bacillus cereus, lipase of porcine pancreas, trypsin, and papain. The inhibitor also showed the ability to decrease membrane-bound phospholipase A1 and A2 activities in intracellular organelles such as plasma membranes, mitochondria, lysosomes, and microsomes. In view of these facts, it was concluded that the plasma inhibitor is specific for phospholipase A.     

Oligomers of prostaglandin B1 inhibit in vitro phospholipase A2 activity

Oligomers of prostaglandin B1 inhibited phospholipase A2 extracted from human neutrophils in a dose-dependent manner (IC50 = 5 microM), while the monomer was not inhibitory at concentrations of 10 microM or less. The inhibitory activity of PGB1 oligomers increased with increasing polymer size; PGB dimer had approximately one-half the maximal inhibitory activity of PGBx, while a trimer was almost as inhibitory as a tetramer and PGBx (n = 6). PGBx as an oil or as a water-soluble sodium-salt-inhibited Ca2(+)-dependent phospholipase A2 from snake venom, bovine pancreas, human neutrophil and platelet, human synovial fluid, and human sperm with IC50 values ranging from 0.5-7.5 microM. Inhibition was independent of added Ca2+ and was independent of substrate phospholipid concentration. Interaction of purified snake venom phospholipase A2 (Naja mocambique) with PGBx resulted in dose-dependent quenching of the enzyme's tryptophan fluorescence; 50% quench was noted with a molar ratio of PGBx/enzyme of 1.5. Inhibition of phospholipase A2 activity by PGBx was relieved in a dose-dependent manner by either defatted or untreated bovine serum albumin. PGBx is a potent in vitro inhibitor of a wide spectrum of phospholipases A2, and as illustrated in the accompanying paper, has profound inhibitory effects on arachidonic acid mobilization in human neutrophils and vascular endothelial cells. Modulation of cellular and extracellular phospholipases A2, and the bioactive transmitters generated by this catalytic event, may be a basic mechanism by which oligomers of prostaglandin B1 exert their reported membrane-protective effects.     

Identification and purification of a very high affinity binding protein for toxic phospholipases A2 in skeletal muscle

Oxyuranus scutellatus toxin 1 (OS1) and toxin 2 (OS2) are two monochain phospholipases A2 isolated from the venom of Taipan. Their iodinated derivatives have been used to characterize phospholipase A2 receptors on rabbit skeletal muscle cells in culture. Both ligands recognize one family of binding sites on myotube membranes with a Bmax of 1.9 to 2.2 pmol/mg of protein and dissociation constant values of 7.4 pM for 125I-OS2 and 38 pM for 125I-OS1. Other snake venom phospholipases A2 are able to inhibit 125I-OS2 binding to the muscle receptor. Competition experiments with these unlabeled phospholipases A2 define a pharmacological profile of the muscle receptor very different from the previously described pharmacological profile of the neuronal phospholipase A2 receptors. The number of 125I-OS2 receptors in skeletal muscle cells increases during in vitro cell maturation but there is no clear relation between the increase of Bmax and the fusion of myoblasts into myotubes. The phospholipase A2 binding protein from myotubes has been identified both by cross-linking experiments and by purification studies. It is composed of only one subunit of Mr 180,000.     

Monoclonal antibodies against rat platelet phospholipase A2

Monoclonal antibodies which bind specifically to rat platelet phospholipase A2 have been raised. None of them bound to exocrine phospholipase A2 derived from pancreas or snake venom. All antibodies recognized the conformational structure of rat platelet phospholipase A2 supported by intramolecular disulfide bonds, since the reactivity between the antibodies and the enzyme was lost in the presence of 2-mercaptoethanol. One of them, designed MB5.2, inhibited the activity of the platelet phospholipase A2 in a dose-dependent manner. A kinetic study revealed that antibody MB5.2 apparently competed with the substrate for the active site of the enzyme. The other antibodies, designed MD7.1 and ME6.1, inhibited the binding of the enzyme to heparin. The distribution of phospholipases A2 bearing a similar determinant to rat platelet phospholipase A2 was investigated by immunoprecipitation of the enzyme activity or by an immunoblot technique. Among rat tissues, cross-reactivity was observed with phospholipases A2 from spleen, lung, and bone marrow. Extracellular phospholipase A2 detected in the peritoneal cavity of casein-treated rat was also recognized by these antibodies. Furthermore, antibody MD7.1 cross-reacted with rabbit and guinea pig platelet phospholipases A2.     

广西眼镜王蛇毒碱性磷脂酶A_2(PLA_2)的分离纯化及其性质的研究

广西眼镜王蛇毒用羧甲基纤维素CM-52、磷酸纤维素P-11和Sepharose CL-6B柱层析纯化,得到一个在聚丙烯酰胺凝胶电泳上为单一蛋白带,PLA_2的比活性较原蛇毒提高3.6倍,分子量的为13000,由122个氨基酸组成,_pI为8.9,具有良好的热稳定性。从碱性PLA_2对红细胞影响的电镜观察可见,对人的红细胞膜有明显的作用,而对山羊红细胞作用不明显。PLA_2无论对人还是对山羊、兔和豚鼠红细胞电泳速度都有明显的迟缓作用。