Stereochemistry of hydrolysis by snake venom phosphodiesterase.

[18O]Adenosine 5'-O-phosphorothioate-O-p-nitrophenyl ester was prepared by saponification of the bis (-O,O-p-nitrophenyl ester) with K18OH. Only the diastereoisomer with the Rp configuration si a substrate for snake venom phosphodiesterase. The asymmetrically labeled [18O]adenosine 5'-O-phosphorothioate formed in this reaction was converted enzymatically to [18O]adenosine 5'-(1-thiodiphosphate) with the Sp configuration. The position of the 18O label, either bridging [1,2-mu-18O] or nonbridging [1-18O] was then determined. The results show that the reaction catalyzed by snake venom phosphodiesterase takes place with retention of configuration at phosphorus. This indicates that the hydrolysis proceeds via a covalent nucleotide enzyme intermediate.     

Kinetics of the hydrolysis of monodispersed dihexanoyllecithin catalyzed by the phospholipase A2 from Agkistrodon halys blomhoffii venom

The hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by the phospholipase A2 from the venom of Agkistrodon halys blomhoffii, was studied at 25 degrees C and the ionic strength of 0.1 in the presence of 3-33.3 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micelle concentration (cmc), were analyzed according to the Michaelis-Menten equation. The pH-dependence curve of the Km value exhibited only one transition below pH 8. The analytical results indicated that the pK value of 6.30 of an ionizable group changed to 6.54 on the binding of the monodispersed substrate. This ionizable group was assigned as the alpha-amino group on the basis of its pK value, which had been determined from the pH dependence of the binding constant of monodispersed n-dodecylphosphorylcholine (n-C12PC) (Ikeda and Samejima (1981) J. Biochem. 90, 799-804, and Haruki et al. (1986) J. Biochem. 99, 99-109). The pH-dependence curve of the kcat value exhibited two transitions, below pH 6.5 and above pH 9.5. The analytical results indicated the participation of two ionizable groups with pK values of 5.55 and 10.50. Deprotonation of the former and protonation of the latter group were found to be essential for the catalysis. The former ionizable group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Ikeda et al. (1981) J. Biochem. 90, 1125-1130).(ABSTRACT TRUNCATED AT 250 WORDS)     

Epitope mapping of snake venom phospholipases A2 with pseudexin monoclonal antibodies

Fifteen different monoclonal antibodies, developed against a pseudexin A, B, and C mixture, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin B were synthesized on alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies recognized linear epitopes of pseudexin A, pseudexin B, and also nonidentical sequences found in other phospholipases A₂ (PLA₂s) as determined by enzyme-linked immunosorbent assays. Three antibodies recognized a highly conserved site important in calcium binding and the interlocking of dimeric forms of PLA₂. Antibodies neutralizing lethal or enzymatic effects of PLA₂ did not recognize linear epitopes.     

Characterization of phospholipases A2 of Naja melanoleuca snake venom modified at the N-terminal region

In phospholipase A2 from Naja melanoleuca snake venom all four lysines were converted into the epsilon-amidinated derivatives without reaction of the alpha-amino group. The amidinated phospholipase (AMPA) showed high enzymatic activity. Starting from AMPA, chemical modification reactions were carried out at the alpha-amino function. This group was blocked with a tert-butyloxycarbonyl or a phenylthiocarbamyl group. Furthermore the polypeptide chain was shortened by one residue by removing the N-terminal asparagine, resulting in the formation of des-Asn1-AMPA. The native enzyme was shortened by eight residues by cyanogen bromide cleavage at the single methionine residue. Although all modified proteins show a reduced affinity for monomeric lipids, they are easily saturated with micellar substrate analogs. Whereas the removal of the N-terminal octapeptide abolished all enzymatic activity the other modified enzymes possess a low (1%), but measurable enzymatic activity. It is concluded that chemical modifications in the N-terminal region give rise to a distortion of the active site, thus reducing the activity of the lipid-bound enzyme.     

Kinetics of the hydrolysis of monodispersed dihexanoyllecithin catalyzed by a cobra (Naja naja atra) venom phospholipase A2

The hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by a cobra (Naja naja atra) venom phospholipase A2, was studied at 25 degrees C ionic strength 0.1 in the presence of 3-10 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micellar concentration (cmc), were analyzed according to the Michaelis-Menten equation. The Km value was practically independent of pH (between pH 6.75 and 10.30). This finding was consistent with the result of a direct binding study on monodispersed n-alkylphosphorylcholines (Teshima et al. (1981) J. Biochem. 89, 1163-1174). The hydrolysis of the substrate was competitively inhibited by the presence of monodispersed n-dodecylphosphorylcholine (n-C12PC). These results indicated that the substrate and n-C12PC compete for the same site on the enzyme molecule. The pH dependence curve of the kinetic parameter, kcat/Km, exhibited three transitions, below pH 8, between pH 8 and 9.5, and above pH 10. The analysis indicated the participation of three ionizable groups with pK values of 7.25, 8.50, and 10.4. The deprotonation of the first group and the protonation of the third group were found to be essential for the catalysis. The first group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Teshima et al. (1981) J. Biochem. 89, 13-20).(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of two phospholipases A2 purified from Bothrops alternatus snake venom on macrophages

The in vitro effects of BaltTX-I, a catalytically inactive Lys49 variant of phospholipase A₂ (PLA₂), and BaltTX-II, an Asp49 catalytically active PLA₂ isolated from Bothrops alternatus snake venom, on thioglycollate-elicited macrophages (TG-macrophages) were investigated. At non-cytotoxic concentrations, the secretory PLA₂ BaltTX-I but not BaltTX-II stimulated complement receptor-mediated phagocytosis. Pharmacological treatment of TG-macrophages with staurosporine, a protein kinase C (PKC) inhibitor, showed that this kinase is involved in the increase of serum-opsonized zymosan phagocytosis induced by BaltTX-I but not BaltTX-II secretory PLA₂, suggesting that PKC may be involved in the stimulatory effect of this toxin in serum-opsonized zymosan phagocytosis. Moreover, BaltTX-I and -II induced superoxide production by TG-macrophages. This superoxide production stimulated by both PLA₂s was abolished after treatment of cells with staurosporine, indicating that PKC is an important signaling pathway for the production of this radical. Our experiments showed that, at non-cytotoxic concentrations, BaltTX-I may upregulate phagocytosis via complement receptors, and that both toxins upregulated the respiratory burst in TG-macrophages.     

Kinetics of erythrocyte lysis by snake venom cardiotoxins

Hemolysis of guinea pig erythrocytes by snake venom cardiotoxins was investigated with a semi-automatic method based on light-scattering changes of erythrocyte suspensions at 700 nm which are directly related to hemoglobin release. Small amounts of phospholipase-free cardiotoxin (<100 μg) could be conveniently and rapidly assayed with the high reproducibility in a recording spectrophotometer, and reliable kinetic data were accumulated.Cardiotoxins from two different genera (Hemachatus haemachates and Naja mossambica mossambica) displayed virtually identical hemolytic properties. Hemolysis increased linearly with time, in contrast with a sigmoidal pattern when phospholipase was present as an impurity. Low concentrations of Ca²⁺ (<1 mM) stimulated cardiotoxin action. A limiting plateau rate of hemolysis reached during concentration dependence experiments in which the level of either cardiotoxin or of erythrocytes was varied, suggested that the interaction of cardiotoxin with erythrocyte membranes is a saturation phenomenon only at a high ratio of cardiotoxin: erythrocytes. No hemolysis was observed with an homologous neurotoxin of S-methylated cardiotoxin, providing evidence for specificity. The linear Arrhenius plots obtained for the temperature dependence of cardiotoxin-induced hemolysis strengthened the conclusion that its action involves more than a detergent-like effect on membrane phospholipids.     

Role of Ca2+ in the substrate binding and catalytic functions of snake venom phospholipases A2

Phospholipases A2 are classified into two groups, I and II, according to differences in the polypeptide-chain length and the intramolecular-disulfide bondings. The effects of Ca2+ on the kinetic parameters for the hydrolysis of monodispersed and micellar phosphatidylcholines, catalyzed by a cobra (Naja naja atra) enzyme (Group I) and by mamushi (Agkistrodon halys blomhoffii) and habu (Trimeresurus flavoviridis) enzymes (Group II), were studied by the pH-statassay method at 25 degrees C, pH 8.0-8.2, and ionic strength 0.1-0.2. The results were compared with those reported for the other Group I and II enzymes. The Ca2+ binding was clearly shown to be essential for the catalysis of all the phospholipases A2. However, the substrate binding to Group I enzymes was found to be independent of the Ca2+ binding. On the other hand, the substrate binding to Group II enzymes was facilitated more than 10 times by the binding of Ca2+ to the enzymes. This was interpreted in terms of conformation changes of the peptide loop of residues 26 to 44 accompanying the Ca2+ binding. The latter result, but not the former, seems compatible with the hypothesis for interpreting the catalytic mechanism of phospholipases A2 that an intermediate complex should be stabilized by the coordination of the bound Ca2+ ion with the phosphoryl group and the carbonyl oxygen atom of the ester bond at the sn-2 position of the bound substrate molecule [Verheij et al. (1980) Biochemistry 19, 743-750 and (1981) Rev. Physiol. Biochem. Pharmacol. 91, 91-203]. According to the similarity in the primary and tertiary structures of the active sites of both types of enzymes [Renetseder et al. (1985) J. Biol. Chem. 260, 11627-11634], it is supposed that similar intermediate complexes may occur even for Group I enzymes, at least in the transition state of the productive complexes.     

Kinetics of the hydrolysis of monodispersed and micellar phosphatidylcholines catalyzed by a phospholipase C from Bacillus cereus

The phosphatidylcholine-hydrolyzing phospholipase C, so-called "phospholipase C" (PLC), was isolated from the culture of Bacillus cereus strain IAM 1208. The amino-acid composition and partial N-terminal sequence of the purified enzyme were in good agreement with those expected from the nucleotide sequence for a PLC of strain ATCC 10987 [Johansen et al. (1988) Gene 65, 293-304]. The chain-length dependence of kinetic parameters for the PLC-catalyzed hydrolysis of monodispersed short-chain phosphatidylcholines (diCNPC, N = 3-6) was studied by a pH-stat assay method at 25 degrees C, pH 8.0, and ionic strength 0.2 in the presence of saturating amounts of Zn2+ (0.1 mM). The result was compared with those for snake venom phospholipases A2 [Teshima et al. (1989) J. Biochem. 106, 518-527]. It was found that the interaction of the PLC with the head group of the substrate molecule is very important for the binding. The pH dependences of kinetic parameters for the hydrolysis of monodispersed diC5PC and mixed micelles of diC16PC with Triton X-100 were also studied under the same conditions. An ionizable group, whose pK value is perturbed from 7.77 to 8.30 by substrate binding, was found to be essential to the catalysis. This group was tentatively assigned to His 14 on the basis of the results on X-ray crystallographic and chemical modification studies [Hough et al. (1989) Nature 338, 357-360 and Little (1977) Biochem. J. 167, 399-404].(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereochemical course of the reaction catalyzed by 5'-nucleotide phosphodiesterase from snake venom.

The hydrolysis reaction of ATP alpha S by snake venom phosphodiesterase is highly specific for the B diastereomer and proceeds with 88% retention of configuration at phosphorus. Since this enzyme also catalyzes the hydrolysis of the S enantimoer of O-p-nitrophenyl phenylphosphonothioate, the absolute configuration at A alpha of ATP alpha S (B) is assigned as the R configuration provided the two substrates are processed identically. A mechanism for the hydrolysis reactions catalzyed by the venom phosphodiesterase involving at least a single covalent phosphoryl-enzyme intermediate is in accord with this result.     

Kinetics of alpha-chymotrypsin catalyzed hydrolysis in equilibrium. II. Comparison of ester and amide substrates

Kinetic of the alpha-chymotrypsin catalyzed reversible hydrolytic reaction of methyl N-acetyl-L-phenylalaninate and N-acetyl-L-phenylalanylglycinamide at pH 5.5 and equilibrium conditions has been studied. The rates of the labeled reaction products incorporated into the substrate a different methanol concentrations shows that the reaction proceeds by a compulsory mechanism with the formation of N-acetyl-L-phenylalanine-alpha-chymotrypsin complex. For the amide substrate the data obtained are also in agreement with the compulsory mechanism of its hydrolysis. Equilibrium kinetics of ester and amide substrates hydrolysis has been compared.     

The stereochemical course of hydrolysis catalysed by snake venom 5''-nucleotide phosphodiesterase.

Adenosine 5'-(S)-[16O,17O,18O]phosphate was pyrophosphorylated by the combined action of adenylate kinase and pyruvate kinase. The isotopomers of adenosine 5'-[alpha-16O,17O,18O]triphosphate were hydrolysed by venom 5'-nucleotide phosphodiesterase (Crotalus adamanteus) in H2(17)O. Analysis by 31P nuclear magnetic resonance spectroscopy of the resulting adenosine 5'-[16O,17O,18O]phosphate, after cyclization and esterification, showed that the hydrolysis occurs with retention of configuration at phosphorus. The most likely explanation of this observation is that the enzymic hydrolysis involves a double displacement at phosphorus with a covalent nucleotidyl--enzyme intermediate on the reaction pathway.     

Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom

Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.     

Isolation and characterization of basic myotoxic phospholipases A2 from Bothrops godmani (Godman's pit viper) snake venom.

Two basic myotoxic phospholipases A2 were purified to homogeneity from the venom of Bothrops godmani from Costa Rica by ion-exchange chromatography on CM-Sephadex. They have molecular weights of 14,300 (myotoxin I) and 13,400 (myotoxin II) and isoelectric points of 8.2 (myotoxin I) and 8.9 (myotoxin II). They behave as amphiphilic proteins in charge-shift electrophoresis and have similar amino acid compositions. Both toxins induce drastic myotoxic effects when injected in the gastrocnemius muscle of mice and induce release of peroxidase trapped in negatively charged liposomes. In addition, myotoxin I has high phospholipase A2 activity and is anticoagulant at doses higher than 0.3 microgram/ml, whereas myotoxin II has a very low phospholipase A2 activity and exerts anticoagulant effect only at concentrations higher than 50 micrograms/ml. Immunochemical data indicate that both toxins are immunologically related to Bothrops asper myotoxins, although B. godmani myotoxin II gives a stronger cross-reactivity when tested with antisera raised against B. asper myotoxins I and II.     

Epitope mapping of snake venom phospholipases A2 with pseudexin monoclonal antibodies

Fifteen different monoclonal antibodies, developed against a pseudexin A, B, and C mixture, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin B were synthesized on alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies recognized linear epitopes of pseudexin A, pseudexin B, and also nonidentical sequences found in other phospholipases A₂ (PLA₂s) as determined by enzyme-linked immunosorbent assays. Three antibodies recognized a highly conserved site important in calcium binding and the interlocking of dimeric forms of PLA₂. Antibodies neutralizing lethal or enzymatic effects of PLA₂ did not recognize linear epitopes.