Effects of snake venom phospholipase A2 toxins (beta-bungarotoxin, notexin) and enzymes (Naja naja atra, Naja nigricollis) on aminophospholipid asymmetry in rat cerebrocortical synaptosomes.

The effects of snake venom phospholipase A2 (PLA2) toxins (beta-bungarotoxin, notexin) and PLA2 enzymes (Naja nigricollis, Naja naja atra) on aminophospholipid asymmetry in rat cerebrocortical synaptic plasma membranes (SPM) were examined. Incubation of intact synaptosomes with 2 mM 2,4,6-trinitrobenzene sulfonic acid (TNBS) for 40 min, under non-penetrating conditions, followed by SPM isolation, allowed us to calculate the percentage of phosphatidylethanolamine (PE) and phosphatidylserine (PS) in the outer leaflet of the SPM, while incubation with disrupted synaptosomes provided total labeling values with the difference representing labeling of the inner leaflet. We found that 30% of the PE and 2% of the PS were in the outer leaflet, with 54% of the PE and 80% of the PS in the inner leaflet; 16% of the PE and 18% of the PS was inaccessible to TNBS. PLA2 toxins and enzymes increased in a concentration-dependent manner the percentage of PS and, to a lesser extent, the percentage of PE in the outer leaflet of the SPM, due to a redistribution from the inner to the outer leaflet. There was no correlation between the PLA2 enzymatic activities and the increased percentage of PS in the outer leaflet of the SPM induced by the PLA2 toxins and enzymes. Alteration of aminophospholipid asymmetry does not explain the greater presynaptic specificity and potencies of the PLA2 toxins as compared to the PLA2 enzymes, but may be associated with the increased acetylcholine release from synaptosomes induced by both the toxins and enzymes.     

Do chemical modifications dissociate between the enzymatic and pharmacological activities of beta bungarotoxin and notexin?

We have measured enzymatic, hemolytic and anticoagulant activities, lethal potencies and effects on contractions of the phrenic nerve-diaphragm preparation, by chemically modified derivatives of beta bungarotoxin (beta BuTX) and notexin, two presynaptically acting toxins which have PLA2 activity. The following chemical modifications of beta BuTX were tested: alkylation and methylation of histidine 48, alkylation of tryptophan 19, sulfonylation of tyrosine 68, oxidation of methionines 6 and 8, semicarbazide addition under varied conditions to carboxyl groups, varied extents of carbamylation or trinitrophenylation of lysines and guanidination of all lysines with or without trinitrophenylation of the N-terminal asparagine. Only the histidine, tryptophan and tyrosine residues were modified in notexin. The results obtained were compared with those previously obtained using chemically modified derivatives of Naja nigricollis and Naja naja atra PLA2 enzymes which do not have a specific presynaptic site of action. The results with oxidized methionine and lysine-modified derivatives of beta BuTX are supportive of the suggestions of others that the N-terminal region and basic residues away from the enzymatic active region contribute towards the beta type presynaptic neurotoxicity of the PLA2 toxins. Using modified derivatives of beta BuTX and notexin, the dissociations between enzymatic activities and pharmacological properties were not as marked as previously observed with N. nigricollis and N. n. atra PLA2; nevertheless, several dissociations were noted. We conclude that, just as with non-presynaptically acting PLA2 enzymes, some pharmacological actions of presynaptically acting PLA2 toxins may occur independently of phospholipid hydrolysis.     

Comparative effects of phospholipase A2 neurotoxins and enzymes on membrane potential and Na+/K+ ATPase activity in rat brain synaptosomes.

Beta-Bungarotoxin (beta-BuTX) and notexin are phospholipase A2 (PLA2) neurotoxins which cause an irreversible blockade of neurotransmitter release through specific and potent effects at the presynaptic nerve terminal; however, their mechanism of action is uncertain. We examined the effects of beta-BuTX and notexin on Na+/K+ ATPase activity using Sprague-Dawley rat brain synaptosomes in order to determine if alterations in activity might modulate neurotoxin-induced depolarization. Treatment of synaptosomes with 0.05 to 5 nM beta-BuTX, notexin, and Naja naja atra and Naja nigricollis PLA2 (PLA2 enzymes without selective presynaptic actions) caused a dose-dependent depolarization of synaptosomes with no differences being observed between the effects of the PLA2 neurotoxins and enzymes. N. nigricollis PLA2 (0.5 nM; 20 min) slightly stimulated Na+/K+ ATPase activity while beta-BuTX and notexin (0.5 nM: 10 and 20 min) were without effect. With 50 nM concentrations beta-BuTX and notexin stimulated Na+/K+ ATPase activity, while N. nigricollis and N. n. atra PLA2 inhibited activity. The effects on membrane potential and Na+/K+ ATPase were antagonized or blocked by EDTA (10 mM) and bovine serum albumin (1 mg/ml), suggesting that PLA2 enzymatic activity is essential for their effects on membrane potential and Na+/K+ ATPase activity. Following neurotoxin and enzyme pretreatment, we found a biphasic correlation between synaptosomal free fatty acid (FFA) levels and Na+/K+ ATPase activity, where Na+/K+ ATPase is stimulated by low levels of FFA (0.13 to 0.22 mumol/mg protein) and antagonized by FFA levels in excess of 0.34 mumol/mg protein. In contrast there was a linear correlation between the extent of FFA production and membrane depolarization. We propose that the presynaptic depolarizing actions of beta-BuTX and notexin are not mediated through modulation of Na+/K+ ATPase activity and that the changes observed in ATPase activity and possibly membrane potential are directly due to PLA2 enzymatic activity and the production of FFA.     

Inhibitory effect of EDTA.Ca2+ on the hydrolysis of synaptosomal phospholipids by phospholipase A2 toxins and enzymes

Phospholipases A2 (PLA2) are Ca2(+)-dependent enzymes that are inhibited by EDTA; this inhibition would be expected to be reversed by restoring the Ca2+ concentration. By examining the hydrolysis of synaptosomal phospholipids by PLA2 enzymes, Naja naja atra and Naja nigricollis, and by toxins with PLA2 activity, beta-bungarotoxin (beta-BuTX) and notexin, we demonstrated a novel inhibitory action of EDTA manifested in the presence of excess Ca2+. We postulate the formation of an EDTA.Ca2+ complex which inhibits PLA2 activity in a concentration-dependent manner. Synaptosomes in which phospholipids are hydrolyzed by PLA2 have membranal damage expressed by increased acetylcholine (ACh) release and decreased osmotic activity. Addition of EDTA.Ca2+, which inhibits phospholipid hydrolysis, also reversed the PLA2 effect on ACh release, but not its effect on osmotic activity. The inhibition of PLA2 was observed on membranal phospholipids as well as on an artificial substrate of phospholipid-Triton mixed micelles. Moreover, we found that another enzyme, lactate dehydrogenase, was also inhibited. Our results indicate a non-specific inhibition exerted on the enzyme rather than on the substrate.     

Effects of modification of tyrosines 3 and 62 (63) on enzymatic and toxicological properties of phospholipases A2 from Naja nigricollis and Naja naja atra snake venoms

Previously we selectively modified His (48), Arg, Lys, Asp, Glu and Trp residues in the basic phospholipase A2 from Naja nigricollis and the acidic phospholipase A2 from N. n. atra snake venoms. Evidence was obtained for the existence of separate but perhaps overlapping sites responsible, respectively, for their enzymatic and pharmacological properties. We have now modified one or two (Tyr 3, Tyr 62 [63], Tyr 3 + 62 [63]) out of the nine tyrosine residues in these enzymes using p-nitrobenzenesulfonyl fluoride. The derivatives were separated by HPLC, and modified residues determined by amino acid analysis. Enzymatic activity was tested on lecithin--Triton mixed micelles, egg yolk and heart and diaphragm homogenates. The N. nigricollis modified derivatives retained a greater percentage of their enzymatic activities than did the N. n. atra derivatives and also a greater percentage of their activity on natural substrates than on lecithin--Triton mixed micelles. The greatest loss in activity resulted when both tyrosines were modified and the least when tyrosine 3 was modified. Modification of tyrosine 62 of N. nigricollis caused a much greater loss of intraventricular lethal potency than of enzymatic activity, whereas modification of tyrosine 3 of N. n. atra increased lethal potency over six-fold while enzymatic activity decreased about 60%. Examples of dissociation between enzymatic and pharmacological potencies were also noted when hemolytic, anticoagulant and cardiotoxicity on isolated ventricular muscle were measured. The extents of phospholipid hydrolysis were relatively low in brain homogenates, synaptic plasma membranes and heart ventricular muscle. However, they were similar for the native enzymes and all of the tyrosine modified derivatives. These tyrosines do not appear to be part of the enzymatic active site, even though they are thought to be associated with substrate and calcium binding. These results strengthen our earlier conclusion that some pharmacological effects of phospholipase A2 are not due to enzymatic hydrolysis, and that there are separate but perhaps partly overlapping sites for enzymatic and pharmacological activities.     

Immunochemical properties of Naja naja atra (Taiwan cobra) phospholipase A2 using polyclonal and monoclonal antibodies.

The immuno-chemical properties of Naja naja atra phospholipase A2 (NNA-PLA2) were studied by using the chemically modified PLA2 derivatives and the PLA2 homologues toward anti-NNA-PLA2 polyclonal and monoclonal antibodies. Anti-NNA-PLA2 polyclonal antibodies inhibited the enzymatic activity of NNA-PLA2 and Hemachatus haemachatus DE-I by 87% and 68%, respectively. However, the enzymatic activities of Naja nigricollis CMS-9 and notexin were not significantly affected by the polyclonal antibodies. Competitive enzyme immunoassay revealed that the affinity of NNA-PLA2 for polyclonal antibodies was 330-fold higher than that of Hemachatus haemachatus DE-I. Naja nigricollis CMS-9 and notexin failed to inhibit the binding of NNA-PLA2 to polyclonal antibodies. This implies that the epitope(s) of NNA-PLA2 might comprise some substituted residues in the sequence of PLA2 homologues. Three monoclonal antibodies against NNA-PLA2 were prepared by a hybridoma technique. Two of these monoclonal antibodies inhibited the enzymatic activity of NNA-PLA2, but the other did not. Removal of the N-terminal octapeptide affected the epitope interacting with these monoclonal antibodies. Selective modification of tyrosine residues at positions 3 and 63 or lysine residues at positions 6 and 65 induced a substantial reduction in affinity of NNA-PLA2 for polyclonal and monoclonal antibodies. The three monoclonal antibodies failed to recognize PLA2 homologues. The comparison of the sequence of NNA-PLA2 to those of PLA2 homologues showed that most of the amino acid substitutions of PLA2 homologues occur in the spatially nearby region of the N-terminal region and residues at positions 63 and 65.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation of pharmacological and enzymatic activities of snake venom phospholipases A2 by modification of carboxylate groups

The carboxylate groups in an acidic and in a basic phospholipase A2 (PLA2) enzyme, purified, respectively, from Naja naja atra and Naja nigricollis snake venoms, were modified with carbodiimide and semicarbazide. The derivatives modified at pH 3.5 and pH 5.5 had less than 1% (N. nigricollis) or 2% (N. n. atra) residual enzymatic activity, whereas 12-16% enzymatic activity remained following modification at pH 5.5 in the presence of Ca2+. In marked contrast, these derivatives retained variable, but significantly greater, levels of lethal potency, hemolytic and anticoagulant activities, and abilities to block indirectly and directly induced contractions of the diaphragm. By this modification of aspartic and glutamic acid residues we have, for the first time, obtained derivatives of PLA2 which selectively retain greater pharmacological activity relative to enzymatic activity. Previously, we had found that modification of lysine and arginine residues produced derivatives which retain enzymatic activity but show a loss of pharmacological properties. These findings suggest that some pharmacological effects of snake venom PLA2 enzymes are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.     

Phospholipid hydrolysis in serum lipoproteins by a basic phospholipase A2 from Naja nigricollis snake venom and an acidic phospholipase A2 from Naja naja atra snake venom

Apparent Km and Vmax values for PC and PE hydrolysis were determined following exposure of HDL, LDL, and VLDL to a basic phospholipase A2 from N. nigricollis snake venom and an acidic phospholipase A2 from N. nigricollis snake venom and an acidic phospholipase A2 from N. n. atra snake venom. Both enzymes hydrolyzed the lipoprotein phospholipids approximately as fast as they hydrolyzed pure phospholipids in mixed micelles, however, the N. nigricollis enzyme, which has a much stronger anticoagulant effect than the N. n. atra enzyme, had lower apparent Vmax values. These values were highest for phospholipids in VLDL and lowest for HDL, however, the differences between the lipoproteins were relatively small with the N. nigricollis enzyme while the differences were much larger with the N. n. atra enzyme. Fractions of the two enzymes in which varying numbers of lysines were carbamylated showed much larger differences in relative rates of phospholipid hydrolysis in HDL, LDL and VLDL. Triton X-100 eliminates these differences in rates of hydrolysis. These results are discussed in terms of the differences in the organized structure of the lipoprotein classes and in the penetration ability of the phospholipases.     

Effects of beta-bungarotoxin and Naja naja atra snake venom phospholipase A2 on acetylcholine release and choline uptake in synaptosomes

Beta-bungarotoxin is a potent presynaptically acting snake venom toxin that exhibits phospholipase A2 activity. We compared the effects of beta-bungarotoxin and a less toxic snake venom phospholipase A2 on synaptosomal 3H-acetylcholine release and 3H-choline uptake. The purpose of these experiments was to study the mode by which beta-bungarotoxin inhibits 3H-acetylcholine release in this preparation. Under non-depolarizing conditions, both beta-bungarotoxin and Naja naja atra phospholipase A2 stimulated 3H-acetylcholine release from a synaptosomal fraction preloaded with 3H-choline. Beta-bungarotoxin was more potent, but less efficacious, than N. naja atra phospholipase A2. In contrast, both toxins inhibited 3H-acetylcholine release from the synaptosomal fraction incubated with 3H-choline after toxin exposure. In agreement with the results obtained by monitoring acetylcholine release, beta-bungarotoxin and N. naja atra phospholipase A2 appeared to block 3H-choline uptake into the synaptosomal fraction non-competitively. Although the toxins may cause the release of unlabeled choline from synaptosomes, the block of labeled choline uptake could not be explained by decreased specific activity of 3H-choline in the bathing medium. Therefore, beta-bungarotoxin and N. naja atra phospholipase A2 block 3H-acetylcholine release from synaptosomes indirectly by inhibiting the uptake of 3H-choline necessary for 3H-acetylcholine synthesis. In comparing these results using 3H-choline to those in the literature obtained with deuterated choline, there appears to be a difference in apparent toxin action that relates to the type of label (3H or 2H) attached to choline.     

Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A2 from Agkistrodon piscivorus piscivorus snake venom

The basic Lys-49 phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus venom is homologous to the basic Asp-49 PLA2 from the same venom as well as other snake venom PLA2 enzymes. It differs, however, in several respects, most important being replacement of the previously invariant Asp-49 at the calcium binding site by Lys, resulting in a reversed order of addition of calcium and phospholipid, phospholipid binding first. Although the preferences for phospholipid substrates of the two enzymes are identical, the apparent Vmax of the Lys-49 PLA2 was only 1.4 to 3% that of the Asp-49 enzyme. Similarly, the Lys-49 PLA2, compared to the Asp-49 PLA2 had less than 3% of the intraventricular lethal potency and 4% of the anticoagulant activity. The intravenous lethal potency of the Lys-49 enzyme was 20% that of the Asp-49 PLA2 and both had little direct hemolytic activity. In contrast, both enzymes were approximately equipotent on the phrenic nerve-diaphragm preparation and on the isolated ventricle strip of the heart. On the cardiac and neuromuscular preparations, the effects of the Asp-49 PLA2 were accompanied by hydrolysis of phosphatidylcholine and phosphatidylethanolamine, whereas no phospholipid hydrolysis was observed with the Lys-49 PLA2. Evaluation of the present results, along with earlier findings using Asp-49 PLA2 enzymes from Naja nigricollis, Hemachatus haemachatus and Naja naja atra venoms, allows us to conclude that: The A. p. piscivorus Asp-49 PLA2 enzyme resembles the Asp-49 enzymes from N. n. atra and H. haemachatus. In contrast, the A. p. piscivorus Lys-49 PLA2 has much lower enzymatic and anticoagulant activities than the Asp-49 enzymes, but equal cardiotoxic and junctional effects. In contrast to some previous suggestions, basic PLA2 enzymes are not necessarily more toxic than neutral or acidic enzymes. Pharmacological effects upon the heart and phrenic nerve-diaphragm preparation correlate neither with in vitro measurements of PLA2 activity nor with actual levels of phospholipid hydrolysis in the heart or diaphragm. This suggests that PLA2 enzymes exert effects independent of phospholipid hydrolysis.     

Immunological relationships of phospholipase A2 neurotoxins from snake venoms

Polyclonal rabbit antisera were raised against ten snake phospholipase A2 neurotoxins and one snake phospholipase A2 cytotoxin. Immunological cross-reactivities between these toxins, two other snake phospholipase A2 enzymes and pancreatic phospholipase A2 were studied using ELISA technology. All snake phospholipase A2 neurotoxins fell into two main antigenic classes. One antigenic class was composed of all the elapid toxins tested (textilotoxin, taipoxin, notexin, pseudexin and beta-bungarotoxin), the cytotoxic phospholipase A2 from Naja naja atra and pancreatic phospholipase A2. beta-Bungarotoxin seemed to be in an immunological subclass of its own compared to the rest of the elapid toxins. The second antigenic class was comprised of crotalid and viperid phospholipase A2 neurotoxins (crotoxin, concolor toxin, Mojave toxin, vegrandis toxin, ammodytoxin and caudoxin). Our data indicated that the viperid toxins, caudoxin and ammodytoxin, were an immunological subclass apart from the crotalid toxins.     

Role of the N-terminal region in phospholipases A2 from Naja naja atra (Taiwan cobra) and Naja nigricollis (spitting cobra) venoms

The N-terminal alpha-amino groups of two phospholipases A2 (PLA2) from Naja naja atra and Naja nigricollis venoms were selectively modified with trinitrobenzene sulfonic acid, and the modified derivatives were separated by high performance liquid chromatography (HPLC). Trinitrophenylated (TNP) derivatives contained only one TNP group in the alpha-amino group of Asn-1 and showed a marked decrease in enzymatic activity. PLA2 enzymes were cleaved with CNBr, and the N-terminal octapeptide was separated from the large C-terminal fragment by HPLC. Removal of the N-terminal octapeptide from PLA2 enzymes caused a precipitous decrease in enzymatic activity. Enzyme immunoassay and double immunodiffusion revealed that the N-terminal octapeptide is one of the antigenic determinants of PLA2 enzymes. The presence of dihexanoyllecithin influenced the interaction between PLA2 enzymes and 8-anilinonaphthalene sulfonate (ANS), indicating that ANS-binding site of PLA2 enzymes is at or near the substrate binding site. Modification of the N-terminal region perturbed the substrate binding and the binding ability for ANS. The modified derivatives retained their affinity for Ca2+, indicating that the N-terminal region is not involved in Ca2+-binding. A fluorescence study revealed that the alpha-amino group is near Trp residue(s) and that the N-terminal region is important for stabilizing the architectural environment of the Trp residue(s). The results, together with the proposal that Trp residues in PLA2 enzymes are involved in substrate binding, suggest that the N-terminal region of PLA2 enzymes is involved in substrate binding and in maintaining a functional active site.     

Factors influencing the hemolysis of human erythrocytes by cardiotoxins from Naja naja kaouthia and Naja naja atra venoms and a phospholipase A2 with cardiotoxin-like activities from Bungarus fasciatus venom

The effects of red blood cell age and incubation conditions (temperature, divalent cation type and concentration, pH and glucose) on hemolysis induced by cardiotoxin fractions from Naja naja atra and Naja naja kaouthia venoms, a phospholipase A2 with cardiotoxin-like activities from Bungarus fasciatus venom and bee venom phospholipase A2 were examined. Hemolysis by the snake venom toxins was dependent on red blood cell age (aged more susceptible than fresh) and the temperature of incubation (37 degrees C greater than 20 degrees C). Divalent cations at 0.5-2.0 mM enhanced (Ca2+) or slightly decreased (Sr2+, Ba2+) hemolysis due to N. n. kaouthia and N. n. atra toxins, and greatly decreased (Ca2+, Sr2+, Ba2+) hemolysis by these toxins at higher concentrations (5-40 mM). For the B. fasciatus phospholipase A2, Ba2+ and Sr2+ could not fully support hemolysis in any concentration while both low (less than 0.5 mM) and high (greater than 40 mM) Ca2+ enhanced hemolysis. Bee venom phospholipase A2 only induced hemolysis (greater than 10% at greater than 40 mM) at high concentrations of Ca2+. Increasing the pH from 7.5 to 8.5 greatly increased the levels of hemolysis by the snake venom toxins and enzyme. Glucose (5.3 mM) increased hemolysis by the snake venom components at low concentrations of divalent cations (2 mM) and slightly decreased hemolysis at high concentrations (40 mM). Treatment with p-bromophenacyl bromide abolished phospholipase A2 activity of bee venom and B. fasciatus phospholipases, but did not affect hemolytic potency of N. n. kaouthia or B. fasciatus toxins. A similar mechanism, which is independent of phospholipase A2 activity, may be involved in hemolysis by the N. n. kaouthia and N. n. atra cardiotoxins. The B. fasciatus cardiotoxin-like phospholipase A2 appears to have two mechanisms of hemolysis; the first is similar to that of the two typical cardiotoxins and the second appears dependent on phospholipase A2 activity and is only evident at high Ca2+ concentrations.     

Presynaptic toxicity of the histidine-modified, phospholipase A2-inactive, beta-bungarotoxin, crotoxin and notexin

Beta-Bungarotoxin, crotoxin and notexin were treated extensively with p-bromophenacyl bromide in order to modify the histidine group of the active site and to greatly decrease the phospholipase A2(PLA) catalytic activity. They were studied for their residual presynaptic effects on the mouse isolated phrenic nerve-diaphragm and chick biventer cervicis muscle preparations. The modified toxins still had 1.7-5.2% PLA activity, which was inhibited by Sr2+, as is the enzyme activity of the native toxins. The neuromuscular blocking activity of these modified toxins, which was reduced 30-60 fold in the mouse diaphragm, was due to a presynaptic effect, as judged from the unchanged amplitude of m.e.p.p.s in the blocked preparations. In contrast to the native toxins, however, the presynaptic effect of modified beta-bungarotoxin and notexin was neither antagonized by Sr2+ nor accelerated by increasing the rate of nerve stimulation, indicating that the presynaptic effects of the modified beta-bungarotoxin and notexin are not likely to be due to their PLA enzyme activity. The modified toxins retained a much greater fraction of their early presynaptic effects in comparison to their enzymatic and neuromuscular blocking activities, although the time-course of the early effects was delayed. The results indicate that the modified neurotoxins per se have their own presynaptic effects, which are unrelated to the PLA enzyme activities.     

Phospholipase A2 activity-dependent and -independent fusogenic activity of Naja nigricollis CMS-9 on zwitterionic and anionic phospholipid vesicles

CMS-9, a phospholipase A(2) (PLA(2)) from Naja nigricollis venom, induced the death of human breast cancer MCF-7 cells accompanied with the formation of cell clumps without clear boundaries between cells. Annexin V-FITC staining indicated that abundant phosphatidylserine appeared on the outer membrane of MCF-7 cell clumps, implying the possibility that CMS-9 may promote membrane fusion via anionic phospholipids. To validate this proposition, fusogenic activity of CMS-9 on vesicles composed of zwitterionic phospholipid alone or a combination of zwitterionic and anionic phospholipids was examined. Although CMS-9-induced fusion of zwitterionic phospholipid vesicles depended on PLA(2) activity, CMS-9-induced fusion of vesicles containing anionic phospholipids could occur without the involvement of PLA(2) activity. Membrane-damaging activity of CMS-9 was associated with its fusogenicity. Moreover, CMS-9 induced differently membrane leakage and membrane fusion of vesicles with different compositions. Membrane fluidity and binding capability with phospholipid vesicles were not related to the fusogenicity of CMS-9. However, membrane-bound conformation and mode of CMS-9 depended on phospholipid compositions. Collectively, our data suggest that PLA(2) activity-dependent and -independent fusogenicity of CMS-9 are closely related to its membrane-bound modes and targeted membrane compositions.     

Cardiotoxicity of Naja nigricollis phospholipase A2 is not due to alterations in prostaglandin synthesis

The basic phospholipase A2 from Naja nigricollis snake venom is cardiotoxic, causing decreased contractility and arrhythmias at concentrations which induce low levels of phospholipid hydrolysis. Cardiac tissue has a high content of arachidonic acid at the sn-2 position of the major membrane phospholipids, thus increased prostaglandin synthesis might contribute to the cardiotoxic effects of N. nigricollis phospholipase A2. Intracellular action potentials and cardiac contractility were monitored in the isolated right ventricular wall of the rat heart exposed for 1 hr to N. nigricollis phospholipase A2, with or without indomethacin, or to arachidonic acid. The tissues were homogenized, prostaglandins extracted and the 6-keto PGF1 alpha and PGE2 content of the hearts determined. The physiologic effects and prostaglandin content of hearts treated with N. nigricollis phospholipase A2 were not altered by indomethacin nor mimicked by concentrations of arachidonic acid comparable to that present in N. nigricollis phospholipase A2-treated tissue. These results support our previous suggestion that exogenously applied N. nigricollis phospholipase A2 causes cardiotoxic effects by a mechanism that is independent of phospholipid hydrolysis.     

Specificity of action of beta-bungarotoxin on acetylcholine release from synaptosomes.

Presynaptically acting phospholipase A2 (PLA2) neurotoxins such as beta-bungarotoxin (beta-BuTX) specifically modify the release of acetylcholine (ACh) in the periphery, whereas in the central nervous system (CNS) the release of other neurotransmitters such as norepinephrine (NE) and serotonin (5-HT) are also modified. In addition, ACh release in the periphery is modified in a triphasic manner (decrease, then increase, then block), while in the CNS only the increase has been demonstrated. To determine the specificity of the central effects of beta-BuTX we compared the effects of beta-BuTX and N. n. atra PLA2 on the release from rat cerebrocortical synaptosomes of ACh, NE, and 5-HT. We also measured the leakage of lactate dehydrogenase (LDH) in order to determine whether membrane permeablization was responsible for neurotransmitter leakage. Both the PLA2 neurotoxin (5.0 nM) and the non-neurotoxic enzyme (0.5 nM) stimulated the loss of NE and 5-HT, but only at concentrations which induced leakage of LDH. Conversely, beta-BuTX stimulated the release of ACh at a concentration (0.5 nM) which caused no leakage of LDH, while N. n. atra PLA2 (0.5 nM) did not stimulate ACh release. beta-Bungarotoxin thus exerts a specific effect on cholinergic nerve terminals, while the leakage of NE and 5-HT induced by beta-BuTX and N. n. atra PLA2 correlates with membrane disruption due to their PLA2 activities. Within 20 min, 0.5 nM beta-BuTX increased the resting release of ACh and decreased the stimulated release induced by depolarization with 4-aminopyridine, while N. n. atra PLA2 (0.5 nM) did not stimulate ACh release and required 45 min to exert an inhibitory effect. beta-BuTX (5.0 nM) also exerted an inhibitory effect on ACh release stimulated by veratridine, but not by high KCl. It is concluded that in low concentrations that do not disrupt membrane permeability, beta-BuTX acts specifically on cholinergic terminals in rat synaptosomes, where it exerts both stimulatory and inhibitory effects.     

眼镜蛇毒细胞毒素CTX-d的分离纯化及抗肿瘤活性

目的 分离纯化眼镜蛇毒细胞毒素,测定其体内外抗癌作用.方法 应用柱层析及RP-HPLC,从眼镜蛇毒粗毒中分离纯化细胞素素(CTX).体内外抗癌作用利用噻唑兰(MTT)法及对荷瘤小鼠U14瘤的抑瘤作用.结果 分离纯化获得的CTX-d不混有PLA2,在体内外实验中显示明显的抗肿瘤作用.结论 结合阳离子交换层析和RP-HPLC可从眼镜蛇毒中高效分离获得不含PLA2的CTX.     

Comparative study of the cytolytic activity of snake venoms from African spitting cobras (Naja spp., Elapidae) and its neutralization by a polyspecific antivenom

Venoms of several Naja species found in Sub-Saharan Africa, and commonly known as "spitting cobras", induce a predominantly cytotoxic pattern of envenomings that may evolve into tissue necrosis and gangrene. Cytotoxic components of their venoms have been identified as members of the three-finger toxin and phospholipase A(2) protein families. In this study, an in vitro assay using the myogenic cell line C2C12, was utilized to compare the cytolytic activities of venoms from five species of spitting cobras: Naja nigricollis, Naja katiensis, Naja pallida, Naja nubiae, and Naja mossambica. These venoms were strongly cytotoxic, causing a 50% effect at ~1.5 μg/well (15 μg/ml), except for N. katiensis venom, which required nearly twice this amount. Using the cell-based assay, the ability of an equine polyspecific antivenom (EchiTab-Plus-ICP) to neutralize cytotoxicity was assessed. The antivenom completely inhibited the cytotoxic activity of all five venoms, although high antivenom/venom ratios were needed. Neutralization curves displayed the following decreasing order of efficiency: N. nubiae > N. pallida > N. mossambica > N. nigricollis > N. katiensis. Results indicate that neutralizing antibodies toward toxins responsible for this particular effect are present in the antivenom, albeit in low titers. Fucoidan, a natural sulfated polysaccharide known to inhibit the toxic effects of some basic snake venom components, was unable to reduce cytotoxicity of Naja venoms. Results emphasize the need of enhancing the immunogenicity of low molecular mass toxins during antivenom production, as well as to search for useful toxin inhibitors which could complement antivenom therapy.     

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.