Biochemistry and pharmacology of the crotoxin complex. I. Subfractionation and recombination of the crotoxin complex.
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Animals↗
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On the subunit structure of crotoxin: hydrodynamic and shape properties of crotoxin, phospholipase A and crotapotin.
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Animals↗
Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes.
Crotoxin, the presynaptic neurotoxin from Crotalus durissus terrificus, was iodinated and used to demonstrate high affinity, specific binding to guinea-pig (Cavia porcellus) brain synaptosomes and synaptosomal membrane fragments. 125I-crotoxin binding to the membrane fragments displays two binding plateaus, (Kd1 = 4 nM and Kd2 = 87 nM, Bmax1 = 2 and Bmax2 = 4 pmoles/mg membrane protein), but binding to whole synaptosomes revealed only one plateau (Kd = 2 nM and Bmax = 5 pmoles/mg membrane protein). Rosenthal analyses of Scatchard plots yielded similar binding constants in the presence or absence of 0.025% Triton X-100. In addition to equilibrium analyses, kinetic analyses of 125I-crotoxin binding to synaptosomal membrane fragments gave a Kd-value of 3 nM. The Kd value was not significantly changed by the exclusion of added calcium, but the binding site number was lowered. Crotoxin binding was inhibited by the acidic subunit of crotoxin and several presynaptic neurotoxins, which were classified according to their inhibitory properties as, strong (acidic subunit of crotoxin, Mojave toxin, concolor toxin, taipoxin and pseudexin), moderate (ammodytoxin A and textilotoxin), weak (notexin and scutoxin A), very weak (notechis II-5) and non-inhibitory (basic subunit of crotoxin, beta-bungarotoxin, Crotalus atrox and porcine pancreatic phospholipases A2, dendrotoxin, and notechis III-4). Purified acidic subunit of crotoxin, the most potent competitor of crotoxin binding, was somewhat more competitive than intact crotoxin and the other strong inhibitors on a molar basis. Strong, moderate and weak inhibitor groups each differed from the preceding group by requiring about a ten fold increase in concentration to effect a 50% inhibition of crotoxin binding. The weak group was therefore at least two-orders of magnitude less effective than the strong inhibition shown by the acidic subunit of crotoxin. Treatment of synaptosomal membranes with protease K lowered 125I-crotoxin binding, whereas treatment with trypsin did not. Iodinated, phospholipase A2 from C. atrox venom showed no specific binding to whole synaptosomes. Our results demonstrate the presence and describe some of the properties of high affinity, specific binding sites in brain tissue for crotoxin and related presynaptic neurotoxins.
Animals↗
Crotoxin, half-century of investigations on a phospholipase A2 neurotoxin.
Crotoxin, the major toxic component of the South American rattlesnake, Crotalus durissus terrificus, is a neurotoxic phospholipase A2 which exerts its pathophysiological action by blocking the neuromuscular transmission. Crotoxin acts primarily by altering the acetylcholine release from the nerves terminals through a mechanism which has not yet been elucidated. It also acts on postsynaptic membranes by stabilizing the acetylcholine receptor in an inactive conformation very similar to the desensitized state. Crotoxin is made of two dissimilar subunits: a basic and weakly toxic phospholipase A2 component-B, and an acidic and non toxic component-A which does not possess any enzymatic activity. Binding experiments showed that crotoxin subunits dissociate when crotoxin interacts with biological membranes: Component-B binds, whereas component-A appears free in solution. The phospholipase A2 subunit binds in a non saturable, non specific manner, on any kind of biological membranes, whereas in the presence of component-A it interacts only with a limited number of high affinity binding sites present on synaptic membranes but not on erythrocyte membranes. Although the target site (acceptor) of crotoxin has not yet been formally identified, binding experiments carried out with small unilamellar phospholipid vesicles of different compositions indicate that some negatively charged phospholipids like mono and diphosphoinositide phosphates might be an important component of crotoxin acceptor site. Crotoxin is in fact a mixture of several isoforms which have very similar but not identical polypeptide sequences. An individual Crotalus durissus terrificus snake is able to synthesize several crotoxin isoforms which may result of the expression of several isogenes and/or of post-translational events. When compared in quantitative manner, the crotoxin isoforms slightly but significantly differ in their enzymatic and pharmacological properties. Finally, immunochemical investigations carried out with polyclonal antibodies prepared against both crotoxin subunits, showed that non precipitating anti-component-B- antibodies (Fab) inhibit the phospholipase A2 activity of crotoxin and neutralize its lethal potency, suggesting that the catalytic and toxic sites of crotoxin are closely related.
Crotoxin↗
Cross-reactivity and neutralization by rabbit antisera raised against crotoxin, its subunits and two related toxins.
Antisera were raised against intact crotoxin (Crotalus durissus terrificus), Mojave toxin (Crotalus scutulatus scutulatus) and concolor toxin (Crotalus viridis concolor), as well as the subunits of crotoxin. Double immunodiffusion and enzyme-linked immunosorbent assays (ELISA) demonstrated antigenic similarity between these three purified toxins and their subunits. Additionally, when crotoxin antisera were pre-incubated with each of the three toxins before injection, the lethal activity of all were neutralized equally well. Antiserum was considerably more effective in neutralizing crotoxin in vivo when the toxin was injected i.m. than when injected i.v. Antisera against both intact crotoxin and its basic subunit were an order of magnitude more effective than crotoxin acidic subunit antiserum in crotoxin neutralization. Purified phospholipase A2 from Crotalus adamanteus and Crotalus atrox showed weak cross-reactivity with antisera raised against intact crotoxin and its subunits in the ELISA. Our results suggest that crotalid neurotoxins can be detected and neutralized by polyclonal antibodies raised against any intact toxin or basic subunit in this group of homologous toxins.
Animals↗
Preparation of a crotoxin neutralizing monoclonal antibody.
Crotoxin is a heterodimeric protein composed of an acidic and basic subunit from the venom of Crotalus durissus terrificus and is representative of a number of presynaptically acting neurotoxins found in the venom of rattlesnakes. Four different monoclonal antibodies, typed as IgG1 subclass, were raised against the basic subunit of this toxin. One was a potent neutralizing antibody of intact crotoxin, which could neutralize approximately 1.6 moles of purified crotoxin per mole of antibody. The monoclonal antibody enhanced the neutralizing ability of commercial polyvalent crotalid antivenom against the lethality of crude C. d. terrificus venom four-fold. Paradoxically, this monoclonal antibody by itself was ineffective against the lethality of crude C. d. terrificus venom. Using an enzyme-linked immunosorbent assay, we tested various proteins for competitive inhibition of binding of biotinylated-crotoxin to plates coated with the four individual monoclonal antibodies. Concolor toxin, vegrandis toxin, intact crotoxin, Mojave toxin, and the basic subunit of crotoxin showed increasing effectiveness as displacers of crotoxin from the neutralizing monoclonal antibody. None of the monoclonal antibodies reacted with purified phospholipase A2 enzymes from Crotalus atrox or Crotalus adamanteus, nor any of the components present in the crude venoms from four different elapids known to contain presynaptically acting neurotoxins, which show some sequence identity to crotoxin.
Animals↗
Identification of a crotoxin-binding protein in membranes from guinea pig brain by photoaffinity labeling.
Crotoxin is a neurotoxic phospholipase A2 capable of blocking synaptic transmission by inhibiting the release of neurotransmitters. The photoaffinity labeling technique was used to identify the neural membrane molecules involved in the binding of crotoxin. A photoactivatable, radioactive derivative of crotoxin was synthesized by reacting crotoxin with N-hydroxysuccinimidyl-4-azidobenzoate and with Na[125I]. Photoirradiation of synaptosomes from guinea pig brains in the presence of the crotoxin derivative resulted in the formation of a major radioactive conjugate of 100,000 daltons as revealed by autoradiography of a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern. Pretreatment of the synaptosomes with trypsin, Staphylococcus aureus protease, or papain prevented the formation of this conjugate. The conjugate was not detected when plasma membranes from several nonneural tissues replaced the brain synaptosomes. Unmodified crotoxin inhibited the formation of this adduct with an IC50 of about 10(-8)M. Mojave toxin, caudoxin, notexin, Naja naja PLA, and taipoxin also inhibited adduct formation with different potencies, while beta-bungarotoxin and pancreatic PLA were ineffective. We concluded that an 85,000-dalton protein is the major component responsible for the binding of crotoxin to synaptosomal membranes.
Affinity Labels↗
Binding of divalent and trivalent cations with crotoxin and with its phospholipase and its non-catalytic subunits: effects on enzymatic activity and on the interaction of phospholipase component with phospholipids.
We have studied the interaction of divalent and trivalent with a potent phospholipase A(2) neurotoxin, crotoxin, from Crotalus durissus terrificus venom. The pharmacological action of crotoxin requires dissociation of its catalytic subunit (component B) and of its non-enzymatic chaperone subunit (component A), then the binding of the phospholipase subunit to target sites on cellular membranes and finally phospholipid hydrolysis. In this report, we show that the phospholipase A(2) activity of crotoxin and of component B required Ca2+ and that other divalent cations (Sr2+, Cd2+ and Ba2+) and trivalent lanthanide ions are inhibitors. The lowest phospholipase A(2) activity was observed in the presence of Ba2+, which proved to be a competitive inhibitor of Ca2+. The binding of divalent cations and trivalent lanthanide ions to crotoxin and to its subunits has been examined by equilibrium dialysis and by spectrofluorimetric methods. We found that crotoxin binds two divalent cations per mole with different affinities; the site presenting the highest affinity (K(d) in the mM range) in involved in the activation (or inhibition) of the phospholipase A(2) activity and must therefore be located on component B, the other site (K(d) higher than 10 mM) is probably localized on component A and does not play any role in the catalytic activity of crotoxin. We also observed that crotoxin component B binds to vesicular and micellar phospholipids, even in the absence of divalent cations. The affinity of this interaction either does not change or else increases by an order of magnitude in the presence of divalent cations.
Cations, Divalent↗
Effects of crotoxin on autonomic neuromuscular transmission in the guinea-pig myenteric plexus and vas deferens.
The effects of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus on mammalian autonomic neuromuscular transmission, have been investigated. In the longitudinal muscle of the guinea-pig ileum, crotoxin induced a dose-dependent contraction which was followed by relaxation, in spite of the continued presence of the toxin. The contractile response was inhibited by indomethacin, tetrodotoxin, verapamil or nifedipine, but was unaffected by atropine, propranolol, mepyramine or methysergide. In addition, crotoxin caused a presynaptic inhibition of the electrically-evoked twitch of the longitudinal muscle of the guinea-pig ileum. In the guinea-pig vas deferens crotoxin also caused an inhibition of the response to field stimulation. The inhibition was reversible after washing and the preparation remained insensitive to further doses of the toxin. The inhibitory effects of crotoxin were not mediated by noradrenaline and were not due to a non-specific smooth muscle depression, because it was not associated with any reduction in motor responses to acetylcholine, ATP, bradykinin or substance P. Pre-incubation of the guinea-pig vas deferens with indomethacin blocked the inhibitory effects of the toxin. This suggests that the presynaptic activity of crotoxin in the vas deferens might be mediated by prostaglandins.
Animals↗
Immunochemical cross-reactivity of two phospholipase A2 neurotoxins, agkistrodotoxin and crotoxin.
Polyclonal rabbit antisera were raised against the phospholipase A2 neurotoxin agkistrodotoxin (AGTX) from Agkistrodon blomhoffii brevicaudus venom and against the phospholipase A2 subunit (component-B, CB) of crotoxin from Crotalus durissus terrificus venom. Anti-AGTX antibodies cross-reacted strongly with crotoxin and crotoxin-like molecules and more weakly with other phospholipases A2 from the venoms of Viperidae and Crotalidae. On the other hand, anti-CB antibodies cross-reacted with AGTX, and also recognized ammodytoxin A and the phospholipase A2 from Vipera berus venom, but not other phospholipases A2 from Crotalidae and Viperidae. Anti-AGTX and anti-CB antibodies were able to inhibit the phospholipase A2 activity and to neutralize the lethal potency of the homologous and heterologous toxins (AGTX or crotoxin). Immunoaffinity chromatography columns were used to isolate anti-AGTX antibodies which recognized CB (91% of the total anti-AGTX antibodies), and anti-CB antibodies which recognized AGTX (52% of the total anti-CB antibodies). Immunochemical investigations performed with each type of antibody indicated that the majority of AGTX antigenic determinants are present on crotoxin component-B and on phospholipases A2 from Viperidae venoms, and that some of these determinants are involved in the neutralization of lethal potency and in the inhibition of enzymatic activity of AGTX and crotoxin.
Animals↗
Crotoxin, a phospholipase A2 neurotoxin from the South American rattlesnake Crotalus durissus terrificus: purification of several isoforms and comparison of their molecular structure and of their biological activities.
Crotoxin, the major toxin of the venom of the South American rattlesnake Crotalus durissus terrificus is a mixture of several isoforms that differ slightly in their molecular structure. The toxin consists of two nonidentical subunits: a basic and weakly toxic phospholipase A2, component B, and an acidic and nontoxic subunit, component A. In the present investigation, we have used fast-performance liquid chromatography (FPLC) on anionic and cationic exchange columns to purify isoforms of both crotoxin subunits. Two component A isoforms and four component B isoforms were obtained in a homogeneous state, and their purity was verified by isoelectric focusing in polyacrylamide gels. The amino acid composition of the purified component A and component B isoforms was in good agreement with the protein sequences determined previously with mixtures of isoforms. The amino acid compositions indicated that for both crotoxin components the isoforms differed only by the replacement of few amino acid residues. Eight crotoxin complexes have been prepared in a homogeneous state by reassociation of pure component A and component B isoforms. The quantitative comparison of enzymatic and pharmacological properties of the reconstituted crotoxins indicated that the two component A isoforms had identical properties, whereas the four component B isoforms fell in two classes: crotoxin complexes formed with component B isoforms of the first class were enzymatically less active and pharmacologically more potent than those obtained with component B isoforms of the second class.
Amino Acids↗
Binding of crotoxin, a presynaptic phospholipase A2 neurotoxin, to negatively charged phospholipid vesicles.
Crotoxin, isolated from the venom of Crotalus durissus terrificus, is a potent neurotoxin consisting of a basic and weakly toxic phospholipase A2 subunit (component B) and an acidic nonenzymatic subunit (component A). The nontoxic component A enhances the toxicity of the phospholipase subunit by preventing its nonspecific adsorption. The binding of crotoxin and of its subunits to small unilamellar phospholipid vesicles was examined under experimental conditions that prevented any phospholipid hydrolysis. Isolated component B rapidly bound with a low affinity (Kapp in the millimolar range) to zwitterionic phospholipid vesicles and with a high affinity (Kapp of less than 1 microM) to negatively charged phospholipid vesicles. On the other hand, the crotoxin complex did not interact with zwitterionic phospholipid vesicles but dissociated in the presence of negatively charged phospholipid vesicles; the noncatalytic component A was released into solution, whereas component B remained tightly bound to lipid vesicles, with apparent affinity constants from 100 to less than 1 microM, according to the chemical composition of the phospholipids. On binding, crotoxin or its component B caused the leakage of a dye entrapped in vesicles of negatively charged but not of zwitterionic phospholipids. The selective binding of crotoxin suggests that negatively charged phospholipids may constitute a component of the acceptor site of crotoxin on the presynaptic plasma membrane.
Chemical Phenomena↗
Early induction by crotoxin of biphasic frequency changes and giant miniature endplate potentials in frog muscle.
1. Following the addition of crotoxin (250 nM) at the frog neuromuscular junction, there was an initial fall in frequency of miniature endplate potentials (m.e.p.ps), followed by a secondary rise which was characterized by the appearance of large spontaneous potentials (giants, g.m.e.p.ps) and an occasional large potential of the burst type. 2. In the presence of 2-(4-phenylpiperidino)cyclohexanol (AH5183, vesicamol), an inhibitor of vesicular acetylcholine uptake, the frequency of g.m.e.p.ps induced by crotoxin was reduced. 3. The characteristic changes in m.e.p.p. frequency and amplitude distribution were absent with crotoxin in Sr-EGTA Ringer. In the presence of high concentrations of Mn (3.6 or 5.4 mM with 0.9 mM Ca), the crotoxin-induced initial fall and the onset of the secondary rise in m.e.p.p. and g.m.e.p.p. frequencies were slower. The timing of these phases was unaffected by Ca concentrations ranging from 6.3 to 0.9 mM. 4. High concentrations of Mn ions partially inhibited the phospholipase A2 activity of crotoxin on artificial phospholipid membranes. This also supports the involvement of the Ca-dependent phospholipase A2 subunit in both phases of the physiological action of the toxin. 5. G.m.e.p.ps were associated with a moderate increase in m.e.p.p. frequency (2-3 s-1) and were of a time-course similar to that of m.e.p.ps. They persisted after washing with medium lacking Ca ions and in the presence of Ca-Mn Ringer that blocked evoked responses. 6. It is concluded that crotoxin, acting through its phospholipase A2 subunit, produces specific disturbances of synaptic exocytosis and vesicle formation in the axolemma of the motor nerve terminal which lead to biphasic changes in m.e.p.p. frequency and the onset of large spontaneous potentials.
Animals↗
Effects of Sr2+ and Mg2+ on the phospholipase A and the presynaptic neuromuscular blocking actions of beta-bungarotoxin, crotoxin and taipoxin.
1.beta-Bungarotoxin, crotoxin and taipoxin, presynaptic neurotoxins of snake venom origin, have about the same phospholipid-splitting activities as a much less toxic cobra phospholipase A2 in the presence of Ca2+ and deoxycholate. 2. Sr2+ was a much less effective activator of the enzymes than is Ca2+, the activation by Sr2+ being only 3-6% for beta-bungarotoxin and crotoxin and 12% for taipoxin. 3. Sr2+ also inhibited the Ca2+ -activated enzymes by 80% in the cases of beta-bungarotoxin and crotoxin, but only 16% in the case of taipoxin. 4. Mg2" had no significant effect on beta-bungarotoxin or crotoxin, but activated taipoxin in the presence or absence of Ca2". 5. In Sr2+ -Tyrode lacking Ca2+ all three toxins exhibited the same immediate depression followed by facilitation in the rat and mouse diaphragms, but the final blocking activity was only 3-10% with beta-bungarotoxin and crotoxin and was 30% with taipoxin. 6. In Sr2+ -Tyrode, increasing in the rate of nerve stimulation had less accelerating effect on the development of neuromuscular block than in Ca2+ -Tyrode for any of the toxins. 7. Removal of Mg2+ from Sr2+ -Tyrode did not diminish the potency of taipoxin in blocking neuromuscular transmission, suggesting that enzyme activity at the outer surface of the axolemma does not contribute to the neuromuscular blocking action. 8. All of the results indicate that there are close correlations between the presynaptic activities of these toxins and their phospholipid-splitting activities in the cationic environment prevailing in the axoplasm. Apparently the final blocking effect of these toxins is due to phospholipase A action within the nerve terminal.
Animals↗
Neutralization of lethal potency and inhibition of enzymatic activity of a phospholipase A2 neurotoxin, crotoxin, by non-precipitating antibodies (Fab).
Rabbit antibodies were prepared against both purified catalytic (component-B) and purified non-catalytic (component-A) subunits of crotoxin, the major phospholipase A2 neurotoxin from the South American rattlesnake. They cross-react with crotoxin-like toxins from the venom of several Crotalus species as well as with single-chain phospholipase A2 neurotoxins from Crotalid and Viperid venoms (agkistrodontoxin and ammodytoxin A) but not from Elapid venoms (notexin). Immunological cross-reactions of anti-component-A and anti-component-B sera with crotoxin and with its isolated components A and B showed that component-A exposes determinants of low immunogenicity which are present on component-B, whereas the major antigenic determinants of component-B are not present on component-A. Anti-component-B antibodies, but not anti-component-A antibodies, neutralize the lethal potency of crotoxin and inhibit its enzymatic activity. Furthermore, non-precipitating anti-component-B Fab fragments were as potent as antibodies, indicating that crotoxin neutralization results from the binding of the antibodies to the catalytic subunit, rather than the formation of an immunoprecipitate.
Antibodies↗
The effect of crotoxin on the longitudinal muscle-myenteric plexus preparation of the guinea pig ileum.
The effects of crotoxin, the neurotoxin of the venom of the South American rattlesnake (Crotalus durissus terrificus), was studied by using the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. Crotoxin (0.02-4.0 microM) caused depression of the twitch response of the electrically stimulated preparation. This transitory depression depended on the concentration of crotoxin; since crotoxin diminished the output of acetylcholine, this depression may be due to the inhibition of the release of acetylcholine from the plexus. Crotoxin also induced an early contraction, followed by relaxation; as the contraction was inhibited by aspirin and indomethacin, it may have resulted from the release of prostaglandin. In addition, a late persistent contracture was observed after the early contraction. The contracture was resistant to blockage by muscarinic, histamine and serotonin antagonists, to hexamethonium, a non-depolarizing ganglionic blocking substance and to tetrodotoxin, a sodium channel blocker. The contracture was blocked by an elevated concentration of calcium (10 mM) and by verapamil, a calcium channel blocker.
Acetylcholine↗
Several isoforms of crotoxin are present in individual venoms from the South American rattlesnake Crotalus durissus terrificus.
Crotalus durissus terrificus venoms collected either from individual snakes or from a large number of animals (more than 30) have been fractionated by high performance liquid chromatography on gel-filtration and ion exchange columns. The chromatographic patterns obtained with individual venom samples indicated that each Crotalus durissus terrificus snake synthesizes five to ten different crotoxin isoforms in widely variable relative proportions. Furthermore, the heterogeneity of venom samples collected from a large number of snakes did not appear significantly larger than that observed with venoms obtained from individual snakes. The comparison of the chromatographic patterns that we obtained with the various (individual and pooled) venoms allowed us to identify about 15 crotoxin isoforms, which may result from the expression of isogenes, since two amino acid variants have been reported to occur at several positions in the sequence of crotoxin component B. These observations confirm the existence of numerous molecular isoforms of crotoxin and suggest that an individual Crotalus durissus terrificus snake possesses several genes coding for the various crotoxin isoforms. The heterogeneity of venom samples collected from a large number of animals is explained, in a large measure, by the complexity of the venom obtained from the individual snakes.
Animals↗
Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions.
Addition of crotoxin to the frog neuromuscular junction in either 0.9 mM Ca2+ plus tubocurarine or 0.5 mM Ca2+ Ringer solution produced a triphasic change in the amplitude of nerve-evoked endplate potentials (e.p.p.s) and, with 0.5 mM Ca2+, a biphasic change in miniature endplate potential (m.e.p.p.) frequency. The secondary phase of rising e.p.p. amplitude was associated with an increase in facilitation of e.p.p. amplitude with closely spaced twin impulses; the increase in spontaneous release lagged behind that of evoked release. When a calcium chelator, BAPTA, was loaded into presynaptic nerve terminals to buffer cytosolic free Ca2+, both e.p.p. amplitude and twin-impulse facilitation were increased by crotoxin to a similar extent relative to that in the control without BAPTA. The duration of the increase in twin-impulse facilitation was reduced but the duration of the increase in e.p.p. amplitude was unaffected by BAPTA loading. The presence of BAPTA did not alter the characteristic changes in spontaneous release in response to crotoxin. These results suggest that the augmentation of evoked and spontaneous transmitter release by crotoxin is not primarily due to changes in cytosolic Ca2+. The response time between stimulus and e.p.p. peak was lengthened in all phases due to prolongation of the interval between stimulus and e.p.p. onset and, in the secondary and tertiary phases, slowing of e.p.p. rise-time. The protein kinase C inhibitor, H-7, produced complex changes in e.p.p.s. under control conditions but did not alter the triphasic response characteristic of intoxication. These results suggest that crotoxin initiates a primary disturbance in the phasic release process leading to a series of time-gated changes which include transient facilitation then uncoupling of phasic release and generalized acceleration of spontaneous release.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗