Relationship between catalysis and toxicological properties of three phospholipases A2 from elapid snake venoms

Phospholipases A₂ from three elapid snake venoms were studied in order to determine if differences in toxicity correlate with differences in pattern or level of phospholipid hydrolysis. The comparatively toxic basic phospholipase A₂ isolated from Naja nigricollis venom exhibits a cardiotoxic action following iv administration in mice that is not exhibited by the less toxic neutral phospholipase A₂ from Hemachatus haemachatus venom or the acidic phospholipase A₂ from Naja naja atra venom. This cardiotoxic action correlates with high levels of phosphatidylserine hydrolysis in heart. Levels and patterns of phospholipid hydrolysis in heart, lung and kidney following iv administration suggest that only the N. nigricollis enzyme has the ability to penetrate permeability barriers in the heart. No cardiotoxic effects are seen following intraventricular injection of a lethal dose of the phospholipases A₂. All three phospholipases A₂ (12 μg/ml) abolish the directly and indirectly elicited muscle twitches of the rat phrenic nerve-diaphragm preparation. This block, in normal or altered bathing media, appears to correlate with the level of phospholipid hydrolysis for the N. naja atra enzyme, but not for the N. nigricollis enzyme. These results suggest that N. nigricollis phospholipase A₂ acts by another mechanism in addition to phospholipid hydrolysis.     

Carbamylation with cyanate of basic phospholipase A2 from the venom of Naja nigricollis (Spitting cobra)

The most basic and toxic phospholipase A₂ purified from Naja nigricollis venom was subjected to lysine modification with cyanate at pH 8.0. The carbamylated derivatives were separated by chromatography on a column of SP-Sephadex C-25 and five fractions were obtained. Amino acid analysis showed that the numbers of Lys-residues modified for fractions Fa to Fe were 10, 9.2, 9.0, 8.2 and 7.4 respectively. The pI values decrease with increasing carbamylation, converting the basic enzyme into an acidic protein. It is noteworthy that even after 9 out of 10 Lys-residues had been modified (Fc) and the pI of the enzyme decreased from 10.6 to 4.4, the enzyme still possessed 75% of its enzymatic activity and 51% of its antigenicity. Although both native and Lys-modified N. nigricollis phospholipase A₂ were perturbed by the presence of Ca²⁺, the difference spectra of Lys-modified Fd differ greatly from that of the native enzyme and become similar to that of the acidic and less toxic phospholipase A₂ from N. naja atra venom. At pH 8.0, the effect of Ca²⁺ on the fluorescence emission intensity of 8-anilinonaphthalenesulfonate-Lys-modified Fd complex was different from that of 8-anilinonaphthalenesulfonate-native N. nigricollis phospholipase A₂ complex and was similar to that of 8-anilinonaphthalenesulfonate-_{N. naja atra} phospholipase A₂ complex. It is clear that the different conformational changes induced by Ca²⁺ might be attributable to the charge properties of the enzyme, which depend on the pH of the solution and the pI of the enzyme. These data are discussed in relationship to the decreased lethal potency and pharmacological effects induced by carbamylation (Condreaet al., 1981b).     

Cardiotoxin from Naja naja atra snake venom: A potentiator of platelet aggregation

Cardiotoxin, isolated from Naja naja atra snake venom, potentiates platelet aggregation induced by ADP, thrombin, collagen and venom phospholipase A₂. The malondialdehyde formation caused by ADP, thrombin and venom phospholipase A₂ were also increased in the presence of cardiotoxin. Both potentiation of aggregation and increase in malondialdehyde were blocked by indomethacin or Ca²⁺ (5 mM or 0.05 mM). Cardiotoxin did not potentiate thrombin-induced aggregation of p-bromophenacyl bromide-modified platelets. Thromboxane B₂ formation induced by thrombin or collagen was also increased by cardiotoxin, while that by arachidonate was not affected. As a membrane-active polypeptide, cardiotoxin might augment the Ca²⁺-flux during the activation of the platelet membrane by aggregation inducers and then increase the activation of endogenous phospholipase A₂.     

Central neurotoxicity of cobra neurotoxin,cardiotoxin and phospholipase A2

Central neurotoxicity of cobra neurotoxin (cobrotoxin), cardiotoxin and phospholipase A₂ isolated from Naja naja atra venom was studied in rats and cats by comparing their lethality and death times following central and peripheral applications. On central application, phospholipase A₂ was most toxic, followed by cardiotoxin, with cobrotoxin being least toxic. The time to death was, in general, much longer by central than by peripheral injection. Cardiotoxin, almost immediately after central application, and phospholipase A₂, after a delay, produced recurrent convulsions. Only mild convulsive movements of the head or extremities were observed with cobrotoxin. The respiratory discharge down the phrenic nerve could be recorded even after complete paralysis of respiratory muscles upon central application of cobrotoxin. From these results, it is concluded that phospholipase A₂ and cardiotoxin, rather than the neurotoxin, are responsible for the high toxicity of cobra venom on central application and that the cause of death by the neurotoxin is peripheral in origin even after central application.     

Inhibition of phosphorylation of rat synaptosomal proteins by snake venom phospholipase A2 neurotoxins (β-bungarotoxin, notexin) and enzymes (Naja naja atra, Naja nigricollis)

E. Ueno and P. Rosenberg. Inhibition of phosphorylation of rat synaptosomal proteins by snake venom phospholipase A₂ neurotoxins (β-bungarotoxin, notexin) and enzymes (Naja naja atra, Naja nigricollis). Toxicon28, 1423–1437, 1990.—Some snake venom neurotoxins, such as β-bungarotoxin (β-BuTX) and notexin, which inhibit the release of neurotransmitter at both peripheral and central presynaptic terminals possess phospholipase A₂ activity. In contrast, most snake venom phospholipase A₂ enzymes such as those isolated from Naja naja atra and Naja nigricollis are structurally homologous to these neutrotoxins but do not have any specific or potent presynaptic action although they have higher enzymatic activities than the neurotoxins. In order to investigate the mechanisms of presynaptic action of the snake venom neurotoxins, we studied their effects on phosphorylation of rat brain synaptosomal proteins. It is known that phosphorylation of synapsin I, a neuron specific and synaptic vesicle associated phosphoprotein, increases neurotransmitter release. Incubation of cerebral cortical synaptosomes with ³²P-orthophosphate at 37°C for 30 min, caused significant phosphorylation of a wide mol. wt range of proteins including most markedly those proteins in the mol. wt range (81,000–86,000) of synapsin I. Both snake venom phospholipase A₂ neurotoxins and enzymes (5, 15 and 50 nM) inhibited phosphorylation in a Ca²⁺-dependent manner with the following order of potencies: β-BuTX >N.n. atra phospholipase A₂ ≥ notexin >N. nigricollis phospholipase A₂. Five nanomoles of β-BuTX, which has the lowest phospholipase A₂ activity, inhibited phosphorylation of a wide range of mol. wt proteins (51,000–188,000) by 42–58%. At the same concentration, N.n. atra phospholipase A₂ (which possesses the highest enzymatic activity), notexin and N. nigricollis phospholipase A₂ caused less inhibition than β-BuTX, ranging from 0–40% depending on the agent used. These results indicate that there is no correlation between their potencies in inhibiting phosphorylation and the levels of their phospholipase A₂ activities. An inhibitory activity on phosphorylation may be at least partially responsible for a presynaptically-induced block of neurotransmitter release.     

Chemical modification of lysine and histidine residues in phospholipase A2 from the venom of Naja naja atra (Taiwan cobra)

The major phospholipase A₂ was isolated from Naja naja atra venom by successive chromatography on SP-Sephadex C-25, DEAE-Sephacel, CM-Sephadex C-25 and SP-Sephadex C-25 columns. The homogeneity was verified by disc electrophoresis and the isoelectric point determined to be 5.2. The specific activity was 3400 U/mg protein, and the ld₅₀ 8 mg/kg mouse. The purified phospholipase A₂ was subjected to lysine modification with cyanate at pH 8.0. After suitable periods (3 and 16 hr), the carbamylated derivatives were separated on a column of DEAE-Sephacel and eight fractions were obtained (DE-1 to DE-8). The results of amino acid analysis showed that one to five Lys-residues were modified. Associated with modification of increasing numbers of Lys-residues were progressive decreases in pI values and marked decreases (3 to > 30-fold) in ld₅₀ values. However, the decrease in enzymatic activity was slight and antigenic specificity was unaffected. The results show a clear dissociation between enzymatic activity and lethal toxicity. The enzyme was also subjected to chemical modification with p-bromophenacyl bromide. Alkylation of the only His-47 at the active site of the phospholipase A₂ destroys both catalytic activity and lethal toxicity, whereas the antigenicity remained unchanged. Although all the native, Lys-modified and His-modified phospholipases A₂ were perturbed by the presence of Ca²⁺ and the difference spectra of Lys-modified DE-6 was similar to that of native phospholipase A₂, the difference spectra of His-modified enzyme differed greatly from that of the native enzyme. The emission intensity of 8-anilinonaphthalenesulfonate-enzyme complex was altered by increasing concentrations of Ca²⁺, and different results were observed at different pH values of the buffer solution, indicating that Ca²⁺ causes pH-dependent conformational changes. The Scatchard plots showed only one kind of specific interaction between 8-anilinonaphthalenesulfonate and native or Lys-modified enzyme (DE-6), and the dissociation constant of Lys-modified DE-6 was similar to that of the native enzyme. On the other hand, the His-modified enzyme lost the ability to bind 8-anilinonaphthalenesulfonate.     

Cobra (Naja Naja Atra) Membrane Toxin Isoforms: Structure and Function

Abstract

Two membrane toxins, termed MT-I and MT-II, were purified to HPLC homogeneity from the venom of Naja naja atra. The NH₂-terminal sequences of the two isoforms were determined. When compared with the known sequences of membrane toxins, we concluded they are CTX-I and CTX-III (from Naja naja atra), respectively. Membrane toxins are basic peptides typified by a chain of 60 amino acids long. Their pi is about 10 and Mr is 6,000-7,000. About half of the amino acids are hydrophobic.

There is lytic synergism between membrane toxins and phospholipase A₂. Membrane toxins, which are different from neurotoxins, are capable of depolarizing muscle cells. The toxins are able to kill cancer cells in vitro. Electrocardiograph of cat to which membrane toxin was applied shows magnificent changes. A positive correlation exists between hydrophobicities and activities of the toxins to inhibit protein kinase C (PKC) activity. All the effects are the result of action of the toxins on cell membranes. In addition, structure-activity relationships are investigated with the available comparative data for membrane toxins centering on LD₅₀, erythrocyte lysis, and muscle cell depolarization.     

Effect of carboxylate group modification on enzymatic and cardiotoxic properties of snake venom phospholipases A2

By treating Naja nigricollis and Naja naja atra phospholipase A₂ with carbodiimide and semicarbazide, we obtained derivatives having varied numbers of modified carboxylate groups. When tested on artificial and natural substrates, derivatives of both enzymes with a modified carboxylate group at the active site (Asp-49) retained little enzymatic activity ($\text{1}\text{4}$1 to 10%). However, the derivatives of N. nigricollis also lost most of their lethal potency (5% of native), while those of N. n. atra retained considerable lethality (29%). Caboxyl modification with protection of Asp-49 in N. n. atra enzyme resulted in a derivative with lethal potency equal to or greater than the native enzyme and enzymatic activity which was low on all substrates (12 – 17% of native). Similar protection of Asp-49 at the active site in N. nigricollis enzyme produced a derivative with decreased enzymatic activity on artificial substrate (22% of native) and decreased lethality (17 – 33% of native), but with full enzymatic activity on natural substrates. When tested on electrical and mechanical properties of the isolated perfused heart and the isolated ventricle muscle wall, the derivatives of both enzymes retained considerably more of the cardiotoxic activity than would have been expected based on their residual enzymatic activity. The one exception occurred with the least modified N. nigricollis derivative which had an unaltered Asp-49, this enzyme retained both cardiotoxic activity and full enzymatic activity on natural substrates. The extent of phospholipid hydrolysis following treatment was measured in the isolated heart preparation and in hearts removed from mice following i.v. injection of the phospholipases. Very low levels of phospholipid hydrolysis were observed and no correlation could be made between the extent of hydrolysis and the pharmacological potencies of these enzymes. Modification of the enzymatic active site, whether of Asp-49 in this study of His-48 in prior studies, leads to a large decrease in both enzymatic activity and lethal potency. Asp and Gluresidues outside of the enzymatic site contribute significantly to the lethal potency of the N. nigricollis enzyme and to the enzymatic activity of the N. n. atra enzyme. Based on these and previous data we conclude that changes in isoelectric points are not responsible for altered lethal potencies following chemical modification and that some pharmacological effects of snake venom phospholipases A₂ are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.     

Basic phospholipase A2 from Naja nigricollis snake venom: phospholipid hydrolysis and effects on electrical and contractile activity of the rat heart

To clarify the mechanism of the cardiotoxic action of the basic phospholipase A₂ from Naja nigricollis snake venom, its effects were tested on the rat heart, including hydrolysis of cardiac phospholipids, electrocardiogram, (Na + K)-ATPase activity, resting and action potentials, and cardiac contractility. Its effects contrasted with those of a noncardiotoxic acidic phospholipase from Naja naja atra snake venom. Only the N. nigricollis enzyme exhibited cardiotoxic effects in isolated perfused rat hearts. The cardiotoxic effects occurred with low levels of phospholipid hydrolysis, phosphatidylserine being hydrolyzed to the greatest extent. Although high concentrations of either phospholipase decreased (Na + K)-ATPase activity in vitro, this reaction did not account for the cardiotoxic effects observed in isolated perfused hearts. In rat atrial preparations the cardiotoxic enzyme depolarizes the cells, decreases action potential amplitude and overshoot, decreases time to 50 and 90% repolarization of action potential, and prolongs the latency to initiation of the action potential. Simultaneously recorded contractile activity showed prolonged latency to initiation of contraction, increased time to peak force of contraction, and decreased amplitude of contraction. These effects were mimicked by caffeine and 7.5 mm Ca²⁺ in the bathing medium. The cardiotoxic effects were blocked by Mn²⁺ (Mn²⁺ also reversed cardiotoxic actions) and 4.0 mm Ca²⁺, but not by lanthanum, tetrodotoxin, lidocaine, acetylcholine, or cyanide. Manganese added to rat atrial preparations before N. nigricollis phospholipase decreased hydrolysis of phosphatidylserine. These results suggest that the N. nigricollis enzyme exerts its cardiotoxic action by increasing intracellular Ca²⁺ levels. The cardiotoxic effects correlate with, but may not be related to, phosphatidylserine hydrolysis.     

Isolation and characterization of a toxic phospholipase A2 in the spitting cobra (Naja mossambica mossambica) venom.

N. Martin-Moutot and H. Rochat. Isolation and characterization of a toxic phospholipase A₂ in the spitting cobra (Naja mossambica mossambica) venom. Toxicon17, 127–136, 1979.—A phospholipase A₂ from the spitting cobra (Naja mossambica mossambica) was purified by chromatographic procedures involving gel filtration on Sephadex G-50 and ion exchange on Amberlite IRC-50. The purified enzyme was homogeneous according to physico-chemical criteria as well as chemical purity tests. This protein was characterized by amino acid composition, determination of N- and C- terminal amino acid sequences, isoelectric pH, molecular weight. It exhibits phospholipase activity and toxicity to mice. Positional specificity towards phosphatidylcholine showed that it belongs to the A₂ type. Optimal activity was observed at an alkaline pH and calcium was required for phospholipase activity.     

Purification, characterization, and cDNA cloning of acidic platelet aggregation inhibiting phospholipases A2 from the snake venom of Vipera lebetina (Levantine viper)

Two novel acidic phospholipase A₂s (PLA₂) were isolated by size exclusion chromatography and reversed-phase chromatography from the crude Vipera lebetina venom. The molecular masses of VLPLA₂-1 (13,704 Da) and VLPLA₂-2 (13,683 Da) and their internal tryptic peptides were determined by MALDI-TOF mass-spectrometry. When tested in human platelet-rich plasma, both enzymes showed a potent inhibitory effect on aggregation induced by ADP and collagen. Chemical modification with p-bromophenacylbromide abolished the enzymatic activity of PLA₂; its anti-platelet activity was fully inhibited in case of collagen as inducer and partially inhibited in case of ADP as inducer. The complete cDNAs encoding PLA₂ were cloned from a single venom gland cDNA library. Complete amino acid sequences of the VLPLA₂ were deduced from the cDNA sequences. The full-length cDNA sequences of the VLPLA₂ possess 615 bp and encode an open reading frame of 138 amino acids that include signal peptide (16 amino acids) and mature enzyme (122 amino acids). The VLPLA₂s have significant sequence similarity to many other phospholipase A₂s from snake venoms. The phylogenetic analysis on the basis of the amino acid sequence homology demonstrates that VLPLA₂s grouped with other Asp49 PLA₂s and they appear to share a close evolutionary relationship with the European vipers.     

Dissociation of enzymatic activity from lethality and pharmacological properties by carbamylation of lysines in Naja nigricollis and Naja naja atra snake venom phospholipases A2

Carbamylation of 9 out of 10 lysine residues in the toxic phospholipase A₂ from N. nigricollis venom decreased its lethality at least 8-fold and abolished its direct hemolytic and anticoagulant activities, while the enzymatic activity, as measured on purified substrates, decreased only about 50%. Likewise, carbamylation of 3 out of 5 lysines in the relatively less toxic N. naja atra phospholipase induced detoxification and caused a loss of its blocking activity on the phrenic nerve-diaphragm preparation, while its enzymatic activity on purified substrates was unaltered. Results obtained when 7.4 out of 10 lysines in N. nigricollis phospholipase were carbamylated indicate that basicity is not an absolute requirement for high lethal potency, hemolytic activity or cardiotoxicity.The extent of phospholipid hydrolysis induced in erythrocytes, rabbit plasma, phrenic nerve-diaphragm preparation, brain minces and brain synaptic plasma membranes by incubation with the carbamylated enzymes was in agreement with their enzymatic activities as measured on purified substrates. Levels of phospholipid hydrolysis in heart, lung and kidney of mice given phospholipase intravenously, and in brain synaptic plasma membranes from rats given phospholipase intraventricularly, showed that carbamylated derivatives of N. nigricollis phospholipase A₂ lost their ability to reach and/or hydrolyze substrates in vivo. However, the decrease in in vivo phospholipid hydrolysis did not correlate with the decrease in toxicity since, at comparably low levels of phospholipid hydrolysis, some phospholipases were lethal and others were not. Moreover, when intraventricularly administered, both lethal amounts of the native N. naja atra enzyme and its nonlethal carbamylated derivatives produced equally low hydrolysis of synaptic membrane phospholipids.By means of lysine carbamylation, a dissociation between hydrolytic activity and pharmacological properties of phospholipases A₂ has been achieved. We suggest, therefore, that the toxicity of pure phospholipases is primarily due to a direct effect which does not correlate with levels of phospholipid hydrolysis and that this direct effect is prominent in the relatively toxic phospholipases while it is less manifest in the relatively non-toxic enzymes.     

Amino acid sequences of phospholipases A2 from the venom of an Australian elapid snake (king brown snake, Pseudechis australis)

Two basic phospholipases A2 (Pa-11 and Pa-13) have been isolated from the venom of an Australian elapid snake, Pseudechis australis (king brown snake). The reduced and S-carboxymethylated phospholipases A2 were digested with trypsin and the resulting peptides were purified by a combination of chromatography on a DEAE-cellulose DE-52 column and gel filtration procedures. Eleven main peptides from Pa-11 and 9 peptides from Pa-13 could account for the amino acid compositions of the respective enzyme molecules. The alignment of the tryptic peptides and unelucidated regions of the amino acid sequences of tryptic peptides were established by the analysis of the peptides obtained by chymotryptic and/or Staphylococcal protease digestions. Each phospholipase A2 consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. Although Pa-11 is enzymatically 30-times as active as Pa-13 and highly toxic as compared to Pa-13, they are highly homologous in their amino acid sequences. They are also homologous to the enzymes from mammalian pancreas and the other snake venom phospholipases A2, especially to those from snakes belonging to the subfamilies Acanthophiinae and Laticaudinae.     

Purification and characterization of a novel antinociceptive toxin from Cobra venom (Naja naja atra)

Snake venoms have demonstrated antinociceptive activity, and certain isolated neurotoxins have demonstrated significant analgesia in animal models. Here we report a novel analgesic toxin which was isolated from Naja naja atra and was given the name ‘najanalgesin’. The LD₅₀ of the crude venom and najanalgesin were 0.89 mg/kg and 2.69 mg/kg, respectively. We used the writhing test and hot plate test to evaluate the antinociceptive properties of the crude venom and najanalgesin after intraperitoneal (ip) administration. The analgesic mechanism of najanalgesin was also studied. The response latency time was significantly prolonged in the hot plate test after ip administration of the crude venom of Naja naja atra (0.111-0.445 mg/kg) in a dose-dependent manner. Najanalgesin (1 mg/kg) elicited almost the same antinociceptive effect as that of the crude venom of Naja naja atra at the dose of 0.445 mg/kg and remained for 6 h after intraperitoneal injection, shown by hot plate test. The percentage of increase in the latency time for the venom and the najanalgesin 3 h after drug administration was 96.2% and 112%, respectively. The number of writhes decreased to almost 1/3, 1/6, and 1/12 of the NS (physiological saline) group after intraperitoneal administration of najanalgesin at 0.25, 0.5, and 1.0 mg/kg, respectively. Pretreatment with atropine (1 mg/kg) or naloxone (3 mg/kg) blocked the antinociception of najanalgesin in the hot plate test. Based on the sequence information, najanalgesin is found to be highly homologous with the conventional CTXs (cardiotoxins). To our knowledge, no study had previously reported that a toxin which was homologous with CTXs possessed the antinociceptive activity. Thus, this is the first report that the antinociceptive effect induced by najanalgesin is mediated by cholinergic and opioidergic mechanisms.     

Use of sulphopropyl-sephadex chromatography and disc gel electrophoresis for the fractionation and identification of elapid venoms

The cation exchange resin Sulphopropyl (SP)-Sephadex has been used for the preparative isolation of both neurotoxins and cytotoxins from various Elapid venoms. The chromatographic system described affords high resolution of venom components upon elution with simple linear salt gradients. Cytotoxins from the venom of Naja naja naja (India) and neurotoxins from Naja naja atra (Formosa), Naja naja siamensis and Bungarus multicinctus have been isolated in high yield as pure, physiologically-active materials.The resin has also been applied, together with two different discontinuous gel electrophoresis systems, to the characterization of venoms from ten Asian Naja naja subspecies. All of the venoms studied could be identified by their disc gel acidic and basic protein patterns alone, or by a combination of their SP-Sephadex protein elution profiles and the disc gel patterns of their acidic proteins. These studies indicate that the systems reported could be used not only for the identification of specific venoms but could also serve as a basis for a classification system for snake venoms.     

Isolation and amino acid sequence of caudoxin,a presynaptic acting toxic phospholipase A2 from the venom of the horned puff adder (Bitis caudalis)

A presynaptic acting toxic phospholipase A₂, designated caudoxin, was purified from the venom of Bitis caudalis by a combination of gel filtration and ion-exchange chromatography. The specificity of the enzyme was shown to be of the A₂ type. The enzyme contains 121 amino acid residues in a single chain and is cross-linked by seven disulfide bridges. Application of cyanogen bromide cleavage and digestion with trypsin and chymotrypsin yielded peptides providing the necessary overlaps to complete derivation of the sequence. Structural features of caudoxin in relation to other toxic and non-toxic phospholipases A₂ are discussed.     

Lipid domain formation modulates activities of snake venom phospholipase A2 enzymes

The goal of the present study is to elucidate the effect of lipid domain formation on activities of Naja naja atra and Bungarus multicinctus phospholipase A₂ (PLA₂) enzymes. Sphingomyelin inhibited enzymatic activity and membrane-damaging activity of PLA₂ against egg yolk phosphatidylcholine (EYPC), while cholesterol and cholesterol sulfate abrogated the inhibitory effect of sphingomyelin. The ability of cholesterol and cholesterol sulfate to abolish the inhibitory effect of sphingomyelin was closely related to their capacity to induce domain formation in EYPC/sphingomyelin vesicles. Laurdan fluorescence measurement revealed that membrane packing of EYPC/sphingomyelin vesicles was differently affected by cholesterol and cholesterol sulfate. Unlike cholesterol, cholesterol sulfate was unable to promote domain formation in dipalmitoylphosphatidylcholine (DPPC) vesicles. Cholesterol increased but cholesterol sulfate reduced PLA₂ activity against DPPC. Self-quenching studies and trinitrophenylation of Lys residues revealed that PLA₂ enzymes adopted different membrane-bound mode upon absorption onto the membrane bilayers comprised of different lipid compositions. Collectively, our data indicate that lipid domain formation regulates PLA₂ activity, and suggest that the physical state of membrane bilayers changes the interactive mode of PLA₂ with phospholipids.     

Comparison of the enzymatic and edema-produscing activities of two venom phospholipase A2 enzymes

The edema-producing activity of NNAVPLA₂, an acidic phospholipase A₂ (PLA₂) enzyme from Naja naja atra venom (NNAV), was less potent than that of TMVPLA₂ II, a basic PLA; from Trimeresurus mucrosquamatus venom (TMV). These edema-forming effects were greatly suppressed by pretreatment of rats with diphenhydramine/ methysergide or compound 48/80, which reduced the tissue content of histamine and serotonin. Heparin abolished and suppressed the paw edema caused by protamine and TMVPLA₂ II, respectively, but had no effect on the NNAVPLA₂-induced response. In isolated rat peritoneal mast cells, both PLA₂ concentration dependently induced the release of histamine and β-glueuronidase. Again, TMVPLA₂ II was more potent than NNAVPLA₂. This degranulation effect of mast cells caused by TMVPLA₂ II and protamine was inhibited by heparin, while that caused by NNAVPLA₂ was unaffected. The edema-forming and mast cell degranulation effects were greatly decreased in both PBPB-modified NNAVPLA₂ and PBPB-modified TMVPLA₂ II, in which the catalytic activity of the enzymes was completely lost. PBPB-modified TMVPLA₂ II-induced paw edema was also suppressed by heparin. Furthermore, this edematous response was totally reversed in rat pretreated with aspirin in combination with diphenhydramine and methysergide. These results suggest that the edema-forming effect of PLA₂ is probably dependent on the presence of catalytic, positive charge and pharmacological sites on its molecule.     

Effect of modification of one histidine residue on the enzymatic and pharmacological properties of a toxic phospholipase A2 from Naja nigricollis snake venom and less toxic phospholipases A2 from Hemachatus haemachatus and Naja naja atra snake venoms

Modification of one histidine residue at the active site in the phospholipases A₂ from N. nigricollis, H. haemachatus and N. naja atra snake venoms reduced the enzymatic activity to 0·–0·8% that of the native species, while the Km values for the first two enzymes listed remained practically unchanged. Also unchanged following modification were the characteristic substrate specificities of the three enzymes. While it appears most likely that the residual activity of the modified enzymes originates from contamination by non-modified species, the modified enzyme might have residual activity of its own. The decrease in enzymatic activity was accompanied by a decrease in all other biochemical and physiological parameters studied such as: direct hemolysis (for N. nigricollis phospholipase), ability to hydrolyze red cell phospholipids, intravenous ld₅₀ in mice, intraventricular ld₅₀ and cd₅₀ in rats, brain phospholipid hydrolysis and ability to block action potentials in eel electroplax. The results support the view that catalytic activity is essential for toxic action.     

Complete amino acid sequences of two cardiotoxin-like analogues from Bungarus fasciatus (banded krait) snake venom

Three cardiotoxin-like proteins have been isolated from Bungarus fasciatus venom and the amino acid sequence of the major toxin (toxin VI) have been determined. The amino acid sequences of two other analogues (toxins V-2 and V-3) were investigated. The reduced and S-carboxymethylated toxins were digested with trypsin-TPCK and the resulting tryptic peptides were isolated by fingerprinting technique on paper. The amino acid compositions, N-terminal residues and partial amino acid sequences of some of the tryptic peptides were determined. Thirteen tryptic peptides were aligned by following the order of corresponding fragments of toxin VI. Toxins V-2 and V-3 contained 118 and 117 amino acid residues, respectively, in a single polypeptide chain cross-linked with six pairs of intramolecular disulphide bonds. There are only four (for toxin V-2) and five (for toxin V-3) places of differences in their primary structures when compared with that of the major toxin (toxin VI) of Bungarus fasciatus venom.