Membrane damage by Cerebratulus lacteus cytolysin A-III. Effects of monovalent and divalent cations on A-III hemolytic activity

The effects of monovalent and divalent cations on the hemolytic activity of Cerebratulus lacteus toxin A-III were studied. The activity of cytolysin A-III is remarkably increased in isotonic, low ionic strength buffer, the HC50 (the toxin concentration yielding 50% lysis of a 1% suspension of erythrocytes after 45 min at 37 degrees C) being shifted from 2 micrograms per ml in Tris or phosphate-buffered saline to 20-30 ng per ml in sucrose or mannitol buffered with Hepes, corresponding to a 50-100-fold increase in potency. On the contrary, hemolytic activity decreases progressively as the monovalent cation concentration in the medium increases for Na+, K+, or choline salts. The divalent cations Ca2+ and Zn2+ likewise inhibit the cytolysin A-III activity, but more strongly than do the monovalent cations specified above. Zn2+ at a concentration of 0.3 mM totally abolishes both toxin A-III-dependent hemolysis of human erythrocytes and toxin-induced leakage from liposomes. The observation of similar effects in both natural membranes and artificial bilayers suggests an effect of Zn2+ on the toxin A-III-induced membrane lesion, especially since Zn2+ does not alter binding of the cytolysin. The dose-response curve for toxin A-III exhibits positive cooperativity, with a Hill coefficient of 2 to 3. However, analysis of toxin molecular weight by analytical ultracentrifugation reveals no tendency to aggregate at protein concentrations up to 2 mg per ml. These data are consistent with a post-binding aggregational step which may be affected by the ionic strength of the medium.     

Inhibition of [3H]nitrendipine binding by phospholipase A2

Phospholipase A2 from several sources inhibited [3H]nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC50 values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A2 was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A2 enzymatic activity, shifted the bee venom phospholipase A2 dose-response curve to the left. Pretreatment of brain membranes with bee venom phospholipase A2 (10 ng/ml) for 15 min caused a 2-fold increase in the Kd without changing the Bmax compared with untreated membranes. Extension of the preincubation period to 30 min caused no further increase in the Kd but significantly decreased the Bmax to 71% the value for untreated membranes. [3H]Nitrendipine, preincubated with bee venom phospholipase A2, was recovered and found to be fully active, indicating that the phospholipase A2 did not modify the ligand. It is concluded that phospholipase A2 acts on the membrane at or near the [3H]nitrendipine binding site and that phospholipids play a key role in the interactions of 1,4 dihydropyridine calcium channel antagonists with the dihydropyridine binding site.     

Action of phospholipases on the cytoplasmic membrane of Escherichia coli. Stimulation by melittin

The emission maximum of the single tryptophan residue of melittin was measured in the presence of phosphatidylethanolamine liposomes and Escherichia coli cytoplasmic membranes. In both cases, the fluorescence maximum was shifted to shorter wavelengths indicating a transfer of the indole ring to an apolar environment. E. coli membranes were labelled in position 2 of their phospholipids with [¹⁴C]oleic acid. These membranes were used for measuring the activity of an endogenous phospholipase A₂. A slow hydrolysis is observed, which can be accelerated by adding melittin. The extent of the stimulation depends on the molar ratio of melittin to membrane phospholipid. Under suitable conditions, the initial rate of hydrolysis is six to seven times higher in the presence than in the absence of melittin. The action of the phospholipase A₂ from bee venom is also stimulated by melittin. An identical stimulation was observed with either E. coli membranes or pure phosphatidylethanolamine liposomes as substrate.     

Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers.

The action of purified phospholipases on monomolecular films of various interfacial pressures is compared with the action on erythrocyte membranes. The phospholipases which cannot hyorolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Bacillus cereus, phospholipase A2 from pig pancreas and Crotalus adamanteus and phospholipase D from cabbage, can hydrolyse phospholipid monolayers at pressure below 31 dynes/cm only. The phospholipases which can hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Clostridium welchii phospholipase A2 from Naja naja and bee venom and sphingomyelinase from Staphylococcus aureus, can hydrolyse phospholipid monolayers at pressure above 31 dynes/cm. It is concluded that the lipid packing in the outer monolayer of the erythrocyte membrane is comparable with a lateral surface pressure between 31 and 34.8 dynes/cm.     

Hemolytic effect of phospholipase A2 and orientotoxin from venom of the great hornet, Vespa orientalis

The effect of toxic phospholipase A2 and orientotoxin from the venom of the giant hornet Vespa orientalis on human erythrocytes was studied. It was shown that these venom components are potent hemolytic agents, the efficiency of the latter being by about two orders of magnitude as high as that of phospholipase A2. The hemolytic function of the both components is enhanced in the presence of low concentrations of Ca2+, whereas high concentrations of this cation exert an inhibiting action. Polyvalent cations, in particular, ruthenium red, peptide HR-1, mellitin and cytotoxins Us-1 and Us-5 synergetically increase the hemolytic effect of phospholipase A2. During erythrocyte hemolysis the synergistic effect is manifested upon a combined action of phospholipase A2 and orientotoxin. The combination of these toxins increases the total hemolytic activity and produces a far greater effect than in could be expected in the case of each of these compounds taken separately.     

Melittin-induced alterations in dynamic properties of human red blood cell membranes.

The interaction of bee venom melittin with erythrocyte membrane ghosts has been investigated by means of fluorescence quenching of membrane tryptophan residues, fluorescence polarization and ESR spectroscopy. It has been revealed that melittin induces the disorders in lipid-protein matrix both in the hydrophobic core of bilayer and at the polar/non-polar interface of melittin complexed with erythrocyte membranes. The peptide has been found to act most efficiently at the concentration of the order of 10(-10) mol/mg membrane protein. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased upon melittin binding, which results in a significant increase of the maximum energy transfer efficiency. Significant changes in the fluorescence anisotropy of both 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalenesulphonate bound to erythrocyte ghosts, which have been observed in the presence of melittin and crude venom, indicate membrane lipid bilayer rigidization. The effect of crude honey bee venom has been found to be of similar magnitude as the effect of pure melittin at the concentration of 10(-10) mol/mg membrane protein. Using two lipophilic spin labels, methyl 5-doxylpalmitate and 16-doxylstearic acid, we found that melittin at its increasing concentrations induces a well marked rigidization in the deeper regions of lipid bilayer, whereas the effect of rigidization near the membrane surface maximizes at the melittin concentration of 10(-10) mol/mg (10(-4) mol melittin per mole of membrane phospholipid). The decrease in the ratio hw/hs of maleimide and the rise in relative rotational correlation time (tau c) of iodacetamid spin label, indicate that melittin effectively immobilizes membrane proteins in the plane of the lipid bilayer. We conclude that melittin-induced rigidization of the lipid bilayer may induce a reorganization of lipid assemblies as well as the rearrangements in membrane protein pattern and consequently the alterations in lipid-protein interactions. Thus, the interaction of melittin with erythrocyte membranes is supposed to produce local conformational changes in membranes, which are discussed in the connection with their significance during the synergistic action of melittin and phospholipase of bee venom on red blood cells.     

Modification of phospholipids in erythrocyte membranes by phospholipase D. A fluorescence and ESR spectroscopic study

The conversion of more than 65% of the phospholipids in human erythrocyte membranes to phosphatidyl-methanol and phosphatidic acid by incubation with phospholipase D and methanol increased the dissociation constant of the fluorescence probe ANS compared to untreated membranes, but did not affect the number of binding sites and the limiting fluorescence enhancement at maximal binding (Imax). On the contrary, the cationic fluorescence probe dansylcadaverin showed additional binding sites without a change in Kd and an increase of Imax upon incubation with phospholipase D treated erythrocyte membranes compared to incubations of membranes with the original phospholipid pattern. The characteristic temperature-dependence of the quenching of the membrane protein fluorescence by a membrane-bound nitroxide-labeled stearic acid was not influenced by the modification of the phospholipids. A slight reduction of the order parameter, S, determined by ESR-spectroscopy with the same nitroxide spin-labeled fatty acid incorporated into modified membranes compared to controls was found at 40 degrees C, but not at 25 degrees C. The results were interpreted as an indication of membrane domains that retained their physical properties and lipid composition during the incubation with phospholipase D.     

Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A2 and activation by a 70 kDa protein.

Plasma membranes were isolated from carrot (Daucus carota L.) cells grown in suspension culture and treated with phospholipase A2 from snake or bee venom for 10 min. As a result of this treatment, phosphatidylinositol kinase activity was recovered in the soluble fraction. There was no detectable diacylglycerol kinase or phosphatidylinositol monophosphate kinase activity released from the membranes after the phospholipase A2 treatment. Treating the plasma membranes with phospholipase C or D did not release PI kinase activity. The phospholipase A2-released PI kinase was activated over 2-fold by a heat stable, soluble 70 kDa protein. The partially purified 70 kDa activator increases the Vmax but does not affect the Km of the phospholipase A2-released PI kinase.     

Kinetic aspects of the aggregation of Clostridium perfringens theta-toxin on erythrocyte membranes. A fluorescence energy transfer study

Fluorescence resonance energy transfer was used to monitor aggregation kinetics of the "thiol-activated" cytolysin (perfringolysin O (PFO) or theta-toxin) of Clostridium perfringens on erythrocyte membranes. PFO was labeled with the isothiocyanate derivatives of either fluorescein or tetramethylrhodamine. No detectable change in the hemolytic activity of PFO was detected after modification with either fluorophore at a ratio of 1:2 fluorophore molecules/cytolysin molecule. Fluorescence energy transfer (FET) between the donor (fluorescein.PFO or PFOD) and the acceptor (tetramethylrhodamine.PFO or PFOA) was detected by both quenching of donor fluorescence (520 nm) and by enhancement of acceptor fluorescence (575 nm) upon aggregation of labeled cytolysin molecules. FET was only observed when PFOD and PFOA were incubated in the presence of membranes. FET was not observed when PFOD and PFOA were incubated in a membrane-free solution or when unlabeled toxin was substituted for PFOA. FET was also found to be temperature-dependent. The temperature-dependent rates of change in FET upon mixing labeled toxin with erythrocyte membranes proceeded without a lag phase and displayed an activation energy of 18.7 kcal/mol. At all temperatures aggregation of PFO was virtually complete before the onset of hemolysis, the latter exhibiting a distinct lag phase. The lag period before onset of hemolysis was temperature-dependent and exhibited an activation energy of 23.2 kcal/mol. These results suggest that the aggregation of membrane-associated PFO is necessary to initiate the hemolytic process, and the lag phase which occurs before onset of hemolysis reflects the kinetics of PFO monomer to polymer conversion.     

Differential hydrolysis of erythrocyte and mitochondrial membrane phospholipids by two phospholipase A2 isoenzymes (NK-PLA2-I and NK-PLA2-II) from the venom of the Indian monocled cobra Naja kaouthia

We previously demonstrated that venom from the Indian monocled cobra Naja kaouthia is a rich source of phospholipase A2 enzymes, and we purified and characterized a major PLA2 isoenzyme (NK-PLA2-I) from N. kaouthia venom. In the present study, we report the purification and biochemical characterization of a second PLA2 isoenzyme (NK-PLA2-II) from the same venom. A comparison of the membrane phospholipid hydrolysis patterns by these two PLA2s has revealed that they cause significantly more damage to mitochondrial membranes (NK-PLA2-I > NK-PLA2-II) as compared to erythrocyte membranes due to more efficient binding of the enzymes to mitochondrial membranes. Fatty acid release patterns by these PLA2s from the membrane phospholipid PC-pools indicate that NK-PLA2-I does not discriminate between saturated and unsaturated fatty acids whereas NK-PLA2-II shows a preference for unsaturated fatty acids during the initial phase of attack. The current investigation provides new insight into the molecular arrangement of NK-PLA2-sensitive domains in erythrocyte and mitochondrial membranes and highlights the contribution of polar, but uncharged, amino acids such as serine and cysteine in NK-PLA2 induced membrane damage.     

Binding of Cerebratulus cytolysin A-III to human erythrocyte membranes

Binding of Cerebratulus lacteus cytolysin A-III to intact human erythrocytes and erythrocyte membranes has been investigated. Binding to ghosts is essentially complete within 2.5 min of mixing which is slightly faster than the rate of hemolysis measured with intact cells. Approximately 4 X 10(4) binding sites per cell, exhibiting a K 0.5 of 0.7 microM exist; this compares with 50% hematocrit of about 0.3 microM for A-III. Binding is absent in ghosts extracted with Nonidet P-40, but is unaffected by pretreatment of ghosts with either trypsin or elastase.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

Effect of modification of been venom components on its hemolytic activity and ability to decrease the volume of the human erythrocyte

The crude bee venom was modified by different chemical substances. The inverse correlation was established between increase of the hemolytic activity and decrease of the contractive one under modification of the bee venom by NaOH and KOH. The negligible decrease of the contractive activity occurs in the acid conditions (pH 1,0), while hemolytic one is stationary. Dithiothreitol, mercaptoethanol, urea and high temperature (60-90 degrees C) did not affect activity of the bee venom.     

Activation of phospholipase A2 in rabbit erythrocyte membranes by a novel hemolytic toxin (H-toxin) of Clostridium septicum

A primary effect of a novel H-toxin of Clostridium septicum on the hemolysis of rabbit erythrocytes was shown to be the activation of phospholipase A2 (PLA2) associated with rabbit erythrocyte membranes by 20-fold that of controls. Furthermore, the activation of PLA2 induced by the H-toxin was enhanced in the presence of NAD. The H-toxin itself had no PLA2 activity. On the contrary, the H-toxin bound to palmitic acid at a molar proportion of 1:1 and lost its hemolytic activity. The PLA2 was not activated by the H-toxin bound to palmitic acid. These results suggest that activation of the PLA2 is responsible for development of the hemolytic activity of the H-toxin.     

Inhibitors directed to binding domains in neutrophil elastase

Human neutrophil elastase (HNE) can be inhibited by unsaturated fatty acids, including oleic acid [Ashe, B. M., & Zimmerman, M. (1977) Biochem. Biophys. Res. Commun. 75, 194-199; Cook, L., & Ternai, B. (1988) Biol. Chem. Hoppe-Seyler 369, 627-631], but is not affected by saturated fatty acids. We have shown that the interaction of oleic acid with HNE can be characterized by two apparent inhibitory modes: a high-affinity mode (Ki = 48 +/- 3 nM), resulting in partial noncompetitive inhibition (87% residual activity), and a competitive inhibitory mode of lower affinity (Ki = 16 +/- 1 microM). Binding of oleate in the high-affinity mode induces a blue shift in the endogenous fluorescence arising from the tryptophan residues in HNE. This shift is maximal in the presence of 1 microM oleate; higher concentrations of fatty acid have no further effect on the fluorescence spectrum. The negatively charged fluorescent ester of oleic acid and hydroxypyrenetrisulfonate (HPTSoleate) interacts with HNE at an apparent single site (Ki = 44 +/- 3 nM), resulting in competitive inhibition. A blue shift in the emission maximum of the pyrene fluorescence at 410 nm and a decrease in the ratio of the intensities of the maximum at 388 and 410 nm indicate that upon binding to HNE the environment of the pyrene ring in HPTSoleate becomes more hydrophobic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the membrane surface charge and the potentiation of phospholipase A2 as affected by the cytotoxin from the venom of the Central Asian cobra

The interaction of iodine labelled cytotoxin of the middle-asian cobra venom with erythrocyte membranes has been studied. The pretreatment of erythrocytes by cytotoxin results in the sensitivity to the lytic action of pure phospholipase A2 of the same venom. Using I-labelled serum albumin it has been proved that cytotoxin promotes the binding of acid proteins on the membrane such as phospholipase A2 interacted with it synergetically. In the course of study of cytotoxin effect on the time of adhesion hemispheres of artificial bilayer phospholipide membranes it has been established that due to the basic properties cytotoxin neutralizes the negative charge of the membrane surface. This effect of cytotoxin plays an important role in the potentiation of membrane action of phospholipase A2.     

Effects of fatty acids on activity and calmodulin binding of Ca2+-ATPase of human erythrocyte membranes

Activation and inhibition of Ca2+-ATPase of calmodulin-depleted human erythrocyte membranes by oleic acid and a variety of other fatty acids have been measured. Low concentrations of oleic acid stimulate the enzyme activity, both in the presence and in the absence of calmodulin. Concomitantly, the affinity of the membrane bound enzyme to calmodulin progressively decreases due to competitive interactions of calmodulin and oleic acid with the enzyme. Removal of oleic acid from the membrane by serum albumin extinguishes the activating effect of oleic acid and restores the ability of the enzyme to bind calmodulin with high affinity. High concentrations of oleic acid induce an almost complete and irreversible loss of enzyme activity which cannot be abolished by removal of oleic acid. Despite a complete loss of enzyme activity, binding of calmodulin to membranes is approximately normal after removal of oleic acid. Activities of (Na+ + K+)-ATPase, Mg2+-ATPase and acetylcholine esterase, as well as the total protein content, show no gross changes upon treatment of membranes with increasing amounts of oleic acid, which seems to exclude that membrane solubilisation by oleic acid causes an inactivation of the enzyme.     

Isoform-specific membrane insertion of secretory phospholipase A2 and functional implications

Despite increasing evidence that the membrane-binding mode of interfacial enzymes including the depth of membrane insertion is crucial for their function, the membrane insertion of phospholipase A(2) (PLA(2)) enzymes has not been studied systematically. Here, we analyze the membrane insertion of human group IB PLA(2) (hIBPLA(2)) and compare it with that of a structurally homologous V3W mutant of human group IIA PLA(2) (V3W-hIIAPLA(2)) and with a structurally divergent group III bee venom PLA(2) (bvPLA(2)). Increasing the anionic charge of membranes results in a blue shift of the fluorescence of Trp(3) of hIBPLA(2), a decrease in quenching by acrylamide, and an increase in enzyme activity, reflecting an enhancement in the membrane binding of PLA(2). Fluorescence quenching by brominated lipids indicates significant penetration of Trp(3) into fluid POPC/POPG membranes but little insertion into the solid DPPC/DPPG membranes. Increased membrane fluidity also supports hIBPLA(2) activity, suggesting that membrane insertion of hIBPLA(2) is controlled by membrane fluidity and is necessary for the full activity of the enzyme. Trp fluorescence quenching of the V3W-hIIAPLA(2) and bvPLA(2) by water- and membrane-soluble quenchers indicates substantial membrane insertion of Trp(3) of V3W-hIIAPLA(2), similar to that found for hIBPLA(2), and no insertion of tryptophans of bvPLA(2). Our results provide evidence that (a) structurally similar group IB and IIA PLA(2)s, but not structurally diverse group III PLA(2), significantly penetrate into membranes; (b) membrane insertion is controlled by membrane fluidity and facilitates activation of IB and IIA PLA(2)s; and (c) structurally distinct PLA(2) isoforms may employ different tactics of substrate accession/product release during lipid hydrolysis.     

Design of a modern liposome and bee venom formulation for the traditional VIT-venom immunotherapy

Traditional venom immunotherapy uses injections of whole bee venom in buffer or adsorbed in Al (OH)(3) in an expensive, time-consuming way. New strategies to improve the safety and efficacy of this treatment with a reduction of injections would, therefore, be of general interest. It would improve patient compliance and provide socio-economic benefits. Liposomes have a long tradition in drug delivery because they increase the therapeutic index and avoid drug degradation and secondary effects. However, bee venom melittin (Mel) and phospholipase (PLA(2)) destroy the phospholipid membranes. Our central idea was to inhibit the PLA(2) and Mel activities through histidine alkylation and or tryptophan oxidation (with pbb, para-bromo-phenacyl bromide, and/or NBS- N-bromosuccinimide, respectively) to make their encapsulations possible within stabilized liposomes. We strongly believe that this formulation will be nontoxic but immunogenic. In this paper, we present the whole bee venom conformation characterization during and after chemical modification and after interaction with liposome by ultraviolet, circular dichroism, and fluorescence spectroscopies. The PLA(2) and Mel activities were measured indirectly by changes in turbidity at 400(n m), rhodamine leak-out, and hemolysis. The native whole bee venom (BV) presented 78.06% of alpha-helical content. The alkylation (A-BV) and succynilation (S-BV) of BV increased 0.44 and 0.20% of its alpha-helical content. The double-modified venom (S-A-BV) had a 0.74% increase of alpha-helical content. The BV chemical modification induced another change on protein conformations observed by Trp that became buried with respect to the native whole BV. It was demonstrated that the liposomal membranes must contain pbb (SPC:Cho:pbb, 26:7:1) as a component to protect them from aggregation and/or fusion. The membranes containing pbb maintained the same turbidity (100%) after incubation with modified venom, in contrast with pbb-free membranes that showed a 15% size decrease. This size decrease was interpreted as membrane degradation and was corroborated by a 50% rhodamine leak-out. Another fact that confirmed our interpretation was the observed 100% inhibition of the hemolytic activity after venom modification with pbb and NBS (S-A-BV). When S-A-BV interacted with liposomes, other protein conformational changes were observed and characterized by the increase of 1.93% on S-A-BV alpha-helical content and the presence of tryptophan residues in a more hydrophobic environment. In other words, the S-A-BV interacted with liposomal membranes, but this interaction was not effective to cause aggregation, leak-out, or fusion. A stable formulation composed by S-A-BV encapsulated within liposomes composed by SPC:Cho:pbb, at a ratio of 26:7:1, was devised. Large unilamellar vesicles of 202.5 nm with a negative surface charge (-24.29 mV) encapsulated 95% of S-A-BV. This formulation can, now, be assayed on VIT.     

Hemolytic assay for venom phospholipase A2

A rapid and sensitive spectrophotometric assay for venom phospholipase A₂ based on the hemolysis of guinea pig erythrocytes in the presence of decomplemented serum and cardiotoxin (direct lytic factor) is described. This assay is particularly useful for rapid multisample analyses, such as those used in monitoring chromatography fractions, and is specific for phospholipase A₂ in she presence of other potentially hemolytic venom components. The hemolytic mechanism is shown to be a combination of the action of lysophospholipids liberated from lipoproteins in the serum and the synergistic action of phospholipase A₂ and cardiotoxin on the erythrocyte membrane.