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At least 19 recordsLinked to original sources
PubMed1983

The mechanism of inhibition of phospholipase activity of crotoxin B by crotoxin A.

In the crotoxin complex isolated from Crotalus durissus terrificus venom, the component A inhibits the phospholipase A2 activity of crotoxin B only when the substrate is in the aggregated form, preventing the interaction of the enzyme with lecithin--water interfaces. In contrast, with similar rates of hydrolysis of dihexanoyllecithin monomers, the activity of the crotoxin complex is lower than that of crotoxin B when the substrate is aggregated into micelles. Crotoxin B readily hydrolyses dimyristoyllecithin vesicles, the rate being modulated by the physical state of the phospholipid, suggesting that the enzyme is tightly bound to the interface. With the crotoxin complex the rate of vesicle hydrolysis is much slower (about 1/10 that of crotoxin B) and is little affected by the physical state of the lecithin. Direct binding experiments demonstrate that, in contrast to crotoxin B, the crotoxin complex is unable to interact with lecithin--water interfaces. Together with the free accessibility of the enzyme active site in the crotoxin complex, this evidence suggests that a specific area on the enzyme surface, different from the active site and shielded by crotoxin A in the complex, is responsible for the interaction of crotoxin B with lipid--water interfaces.

Crotalid Venoms↗
PubMed2002

Inhibition of crotoxin phospholipase A(2) activity by manoalide associated with inactivation of crotoxin toxicity and dissociation of the heterodimeric neurotoxic complex.

Crotoxin (CACB complex) is a convulsant heterodimeric neurotoxic phospholipase A(2) (PLA(2)). The role of phospholipid hydrolysis in its epileptogenic properties remains unresolved. We, thus, studied the effect of manoalide (MLD), a PLA(2) inhibitor, on the toxin catalytic activity and its central and peripheral toxicity. Incubation of crotoxin with MLD fully and irreversibly inactivated its enzymatic activity. Interestingly, crotoxin also lost its central neurotoxicity after intracerebroventricular injection and peripheral toxicity after intravenous administration. MLD-treated crotoxin prevented the high affinity binding of [125I]-radiolabeled crotoxin on rat cortex synaptic plasma membranes. Further analysis of MLD-treated crotoxin by non-denaturing PAGE and surface plasmon resonance indicated that the crotoxin complex was dissociated after MLD treatment. Although the loss of MLD-treated crotoxin peripheral neurotoxicity could not be attributed to this dissociation, the presence of free CA subunit might explain the observed competition in binding experiments. In conclusion, the dissociation of the crotoxin complex by MLD, as demonstrated in this study, did not permit to specify the role of the enzymatic activity in crotoxin epileptogenic properties. Other approaches would be required to resolve this question.

Animals↗
PubMed1982

Accessibility of the active site of crotoxin B in the crotoxin complex.

Basic phospholipases A and the crotoxin complex isolated from Crotalus durissus terrificus venom exhibited similar initial reaction rates, time course and degree of hydrolysis of synthetic short chain lecithins in the monomeric state. Although monomeric lecithins seem to promote dissociation of crotoxin up to a certain extent, this cannot explain the high activity observed with the complex. The crotoxin complex is able to bind the non-hydrolyzable analog D-diheptanoyllecithin, as demonstrated by equilibrium gel-filtration, with a dissociation constant of 0.12 mM. This value is similar to the dissociation constant of the crotoxin B-D-diheptanoyllecithin complex (about 0.13 mM), estimated from the protection against enzyme inactivation by p-bromophenacyl bromide, which further supports the free accessibility of the substrate to the enzyme active site in the crotoxin complex. The lack of enzyme inactivation when crotoxin is treated with p-bromophenacyl bromide may be interpreted in terms of the specific requirements of the reagent to react with the enzyme rather than protection of the active site. Crotoxin B inhibition by complex formation with crotoxin A, which is not apparent on monomeric substrates, seems not to involve the active site of the enzyme.

Acetophenones↗
PubMed2003

Crotoxin acceptor protein isolated from Torpedo electric organ: binding properties to crotoxin by surface plasmon resonance.

Crotoxin, a potent neurotoxin from the South American rattlesnake Crotalus durissus terrificus, is a heterodimeric phospholipase A(2) (EC 3.1.1.4), which blocks the release of acetylcholine from peripheral neurons. We previously have suggested the existence of a 48 kDa crotoxin-binding protein in the presynaptic membranes of the electric organ of Torpedo marmorata. Here, we report the purification and characterization of this protein that we called the crotoxin acceptor protein from Torpedo (CAPT). The membranes of electric organs from Torpedo were solubilized with a detergent (4% (w/v) Triton X-100) and CAPT was isolated by affinity chromatography on a crotoxin column. SDS-PAGE showed that the purified protein was homogeneous and cross-linking studies with radioiodinated crotoxin confirmed that it had retained its toxin-binding properties. The purified CAPT has similar molecular mass as crocalbin, a crotoxin-binding protein isolated from porcine brains, yet anti-crocalbin antiserum failed to recognize CAPT. Surface plasmon resonance biosensor technology was used to measure the specific interaction between crotoxin and solubilized CAPT. Using this method, it was possible to follow CAPT throughout the purification procedure. As well, an apparent dissociation constant (K(d)(app)) of 3.4 nM was calculated for the interaction of pure CAPT and crotoxin from the dissociation rate constant (k(off)=1.2 x 10(-2)s(-1)) and the association rate constant (k(on)=3.5 x 10(6)M(-1)s(-1)).

Animals↗
PubMed2001

Effects of chemical modifications of crotoxin B, the phospholipase A(2) subunit of crotoxin from Crotalus durissus terrificus snake venom, on its enzymatic and pharmacological activities.

Crotoxin B, the basic Asp49-PLA(2) subunit from crotoxin, the main component of Crotalus durissus terrificus venom, displays myotoxic, edema-inducing, bactericidal (upon Escherichia coli), liposomal-disrupting and anticoagulant activities. Chemical modifications of His (with 4-bromophenacyl bromide, BPB), Tyr (with 2-nitrobenzenesulphonyl fluoride, NBSF), Trp (with o-nitrophenylsulphenyl chloride, NPSC) and Lys (with acetic anhydride) residues of this protein, in addition to cleavage with cyanogen bromide (CNBr) and inhibition with ethylenediaminetetraacetic acid (EDTA), were carried out in order to study their effects on enzymatic and pharmacological activities. Lethality was reduced after modification of His or Lys residues, as well as after cleavage with CNBr, while enzymatic activity was completely abolished after modification of His or incubation with EDTA. Modification of Lys or Tyr, or cleavage with CNBr, partially reduced enzymatic activity. Anticoagulant activity was modified similarly to enzymatic activity, evidencing the dependency of this pharmacological effect on catalytic activity. Myotoxicity was reduced after modification of His or Lys, as well as after cleavage with CNBr, whereas EDTA reduced this effect to a lesser extent. Bactericidal effect was significantly reduced only after modification of Lys and after cleavage with CNBr. Edema-inducing activity was partially inhibited after treatment with EDTA and strongly reduced after acetylation of Lys residues and cleavage with CNBr, being only partially reduced after His alkylation. On the other hand, liposome disrupting activity was only partially reduced after modification of His and Tyr or after cleavage with CNBr. Modification of Trp residue partially reduced lethality and myotoxicity but did not affect enzymatic or anticoagulant activities. These data indicate that enzymatic activity is relevant for some pharmacological effects induced by crotoxin B (mainly lethal, myotoxic and anticoagulant activities), and also evidence that this subunit of crotoxin displays regions different from the active catalytic site which are involved in some of the toxic and pharmacological effects induced by this phospholipase A(2).

Amino Acid Sequence↗
PubMed2000

Influence of temperature upon effects of crotoxin and gamma-irradiated crotoxin at rat neuromuscular transmission.

The influence of temperature upon the effects of crotoxin (CTX), from Crotalus durissus terrificus venom, and gamma-irradiated (60Co, 2000 Gy) crotoxin (iCTX) was studied in rat neuromuscular transmission 'in vitro'. Indirect twitches were evoked in the phrenic-diaphragm preparation by supramaximal strength pulses with a duration of 0.5 ms and frequency of 0.5 Hz. The phospholipase A(2) (PLA(2)) enzymatic activity of CTX and iCTX was assayed against phosphadityl choline in Triton X-100. At 27 degrees C, CTX (14 microg/ml) did not affect the amplitude of indirectly evoked twitches. However, at 37 degrees C, CTX induced a time-dependent blockade of the neuromuscular transmission that started at 90 min and was completed within 240 min. iCTX (14 microg/ml) was inneffective on the neuromuscular transmission either at 27 or 37 degrees C. The PLA(2) enzymatic activity of CTX at 37 degrees C was 84 and that at 27 degrees C was 27 micromol fatty acid released/min/mg protein, and that of the iCTX at 37 degrees C was 39 micromol fatty acid released/min/mg protein. Thus, it was concluded that the mechanism of detoxification of CTX by gamma radiation at the neuromuscular level relies on the loss of its PLA(2) enzymatic activity.

Animals↗
PubMed1991

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↗
PubMed1997

Effect of gamma irradiation on the behavioral properties of crotoxin.

Crotoxin has been detoxified with gamma radiation in order to improve crotalic antiserum production. Nevertheless, present knowledge of the biological characteristics of irradiated crotoxin is insufficient to propose it as an immunizing agent. Crotoxin is known to increase the emotional state of rats and to decrease their exploratory behavior (Moreira EG, Nascimento N, Rosa GJM, Rogero JR and Vassilieff VS (1996) Brazilian Journal of Medical and Biological Research, 29: 629-632). Therefore, we decided 1) to evaluate the effects of crotoxin in the social interaction test, which has been widely used for the evaluation of anxiogenic drugs, and 2) to determine if irradiated crotoxin induces behavioral alterations similar to those of crotoxin in the social interaction, open-field and hole-board tests. Male Wistar rats (180-220 g) were used. Crotoxin (100, 250, and 500 micrograms/kg) was injected intraperitoneally 2 h before the social interaction test. Similarly, irradiated crotoxin (2000 Gy gamma radiation from a 60Co source) was administered at the doses of 100, 250, and 500 micrograms/kg for the hole-board test, and at the doses of 1000 and 2500 micrograms/kg for the open-field and social interaction tests. ANOVA complemented with the Dunnett test was used for statistical analysis (P < 0.05). Crotoxin decreased the social interaction time(s) at the doses of 100, 250 and 500 micrograms/kg (means +/- SEM) from 51.6 +/- 4.4 to 32.6 +/- 3.7, 28.0 +/- 3.6 and 31.6 +/- 4.4, respectively. Irradiated crotoxin did not induce behavioral alterations. These results indicate that 1) crotoxin may be an anxiogenic compound, and 2) in contrast to crotoxin, irradiated crotoxin was unable to induce behavioral alterations, which makes it a promising compound for the production of crotalic antiserum.

Animals↗
PubMed1989

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↗
PubMed1981

A study on the interaction of crotapotin with crotoxin phospholipase A2, notexin and other presynaptic neurotoxins.

1 Crotapotin, the acidic subunit of crotoxin, greatly potentiated the presynaptic effect of isolated basic phospholipase A (PLA) of crotoxin in both mouse diaphragm and chick biventer cervicis muscles whereas the myotoxic effect was not affected significantly.2 In contrast to crotoxin PLA, the presynaptic effects of notexin and notechis-5, self-active single chain toxins, were antagonized by crotapotin while actions of beta-bungarotoxin were not affected.3 By assaying PLA activity, crotoxin PLA was found to be unstable in physiological salt solution, especially when in contact with muscle, due to massive non-specific binding to and destruction by the muscle.4 The decline of crotoxin PLA was greatly reduced by the presence of crotapotin but not by another acidic protein, volvatoxin A(2), or heparin.5 Notechis-5 was found to be stable even when in the presence of muscles.6 [(3)H]-acetylated crotoxin PLA, which retained about 40% of its original enzyme and presynaptic blocking activities, also bound rapidly to the mouse diaphragm on incubation and this binding was greatly hindered by the simultaneous addition of crotapotin.7 The prevention of binding of crotoxin PLA by crotapotin occurred mostly at those sites where the binding was easily dissociable on washing. No antagonism of binding occurred at the firmly binding site.8 The binding of [(3)H]-acetylated crotapotin was much less than that of crotoxin PLA, and interestingly, the binding was increased by the latter, suggesting that crotapotin may be first bound to the diaphragm together with crotoxin PLA.9 No specific binding at the endplate zone was found either for crotoxin PLA or for crotapotin.10 It is concluded that crotapotin potentiates the presynaptic effect of crotoxin PLA by curtailing its non-specific affinity with muscles, minimizing its dispersal and destruction en route to the nerve terminal, but not by acting as an affinity probe for the nerve terminal.

Animals↗
PubMed1993

Induction of tolerance to crotoxin in mice.

Crotoxin, the major toxic component from the venom of Crotalus durissus terrificus is a potent neurotoxin (LD50, i.p., mice, 0.09 mg/kg) which possesses phospholipase A2 activity and causes a blockade of neuromuscular transmission. In this article, we show that mice injected daily with progressively increasing doses of crotoxin develop tolerance to the lethal action of this toxin. Treated mice tolerated daily doses of crotoxin 20- to 35-fold higher than the original LD50 without the characteristic signs of toxicity. Studies on the isolated phrenic nerve-diaphragm preparation in vitro from control (crotoxin-naive) mice showed that the exposure to 2 to 10 micrograms/ml crotoxin in the bath produced complete transmission blockade in 120 to 150 min. Conversely, the preparations from crotoxin-treated mice required crotoxin concentrations in the range of 17.5 to 100 micrograms/ml to produce complete neuromuscular block, being virtually insensitive during 200 min of exposure to 5 to 10 micrograms/ml crotoxin. Phrenic nerve-diaphragm preparations of control (crotoxin-naive) and crotoxin-treated mice did not show significant differences in sensitivity to the blocking action of carbamylcholine, suggesting that induction of tolerance to crotoxin is likely a presynaptic event.

Animals↗
PubMed1993

Comparison of crotoxin isoforms reveals that stability of the complex plays a major role in its pharmacological action.

Crotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus is a potent neurotoxin consisting of a weakly toxic phospholipase-A2 subunit (CB) and a non-enzymic, non-toxic subunit (CA). Crotoxin complex (CACB) dissociates upon interaction with membranes: CB binds while CA does not. Moreover, CA enhances the toxicity of CB by preventing its non-specific adsorption. Several crotoxin isoforms have been identified. Multiple variants of each subunit give different crotoxin complexes that can be subdivided into two classes: those of high toxicity and low enzymic activity and those of moderate toxicity and a high phospholipase-A2 activity. In this study, we demonstrate that the more-toxic isoforms block neuromuscular transmission of chick biventer cervicis preparations more efficiently than weakly toxic isoforms. The less-toxic crotoxin complexes have the same Km and Vmax as CB alone. In contrast, the more-toxic isoforms are enzymically less active than CB. These differences correlate with the stability of the complexes: less-toxic isoforms are less stable (Kd = 25 nM) and dissociate rapidly (half-life about 1 min), whereas the more-toxic isoforms are more stable (Kd = 4.5 nM) and dissociate more slowly (half-life 10-20 min). The rate of interaction of crotoxin complexes with vesicles of negatively charged phospholipids paralleled the rate of dissociation of the complexes in the absence of vesicles. The differences of pharmacological and biochemical properties of crotoxin isoforms indicate that the stability of crotoxin complexes plays a major role in the synergistic action of crotoxin subunits: a stronger association between the two crotoxin subunits would account for their slower dissociation rate, a weaker enzymic activity, a slower interaction with phosphatidylglycerol vesicles, a faster blockade of neuromuscular transmission and a higher lethal potency.

Animals↗
PubMed1993

Cytotoxicity of crotoxin on murine erythroleukemia cells in vitro.

The cytotoxic effect of crotoxin, a heterodimeric phospholipase A2 from the venom of Crotalus durissus terrificus, was examined on murine erythroleukemia cells in vitro. Crotoxin cytocidal effect on cell growth had an EC50 of approximately 0.1-0.2 microM (3.0-5.0 micrograms/ml) in serum-free medium. Cytotoxicity was independent of cell growth since both quiescent and proliferating cells had similar sensitivities to the toxin. Dissociation of the crotoxin complex and phospholipase A2 activity of its subunit B are required for cytotoxicity, since the covalently linked crotoxin complex or the specific alkylation of the active site on the subunit B abolish the cytotoxic activity on murine erythroleukemia cells. Specific interaction between crotoxin and murine erythroleukemia cells appears to be required since the homologous phospholipase A2 from Crotalus atrox venom, with a higher phospholipase A2 specific activity than crotoxin, was 86-fold less potent than crotoxin. The data in this report show that the cytotoxic effect of crotoxin on murine erythroleukemia cells is consistent with the specific binding of the toxin resulting in cytocidal action mediated by the phospholipase A2 activity of crotoxin subunit B.

Animals↗
PubMed1996

Re-examination of crotoxin-membrane interactions.

The interaction of crotoxin with synaptic membranes from Torpedo marmorata has been re-examined, using radioiodinated toxin. In competition experiments, the 'saturable binding' is usually calculated by subtracting the non-saturable binding, determined in the presence of an excess of unlabelled crotoxin, from total binding. Paradoxically, we observed a notable increase of the 'saturable binding' of 125I-crotoxin, defined in this manner in the presence of a high concentration of certain competitors (e.g. crotoxin subunit B, agkistrodotoxin, ammodytoxin and ammodytin I2). This potentiation effect was analysed by competition and cross-linking experiments. The dissociation of the basic crotoxin subunit CB, which differs markedly from crotoxin in its membrane-binding characteristics, was found to be the main reason for the observed phenomenon. 125I-CB could be released from 125I-crotoxin by exchange with a molecule having sufficient affinity towards CA (e.g. CB or agkistrodotoxin) or it could be dissociated from the specific crotoxin membrane-binding site(s) by a competitor molecule. Our results, therefore, suggest a reinterpretation of previous 125I-crotoxin binding studies.

Animals↗
PubMed1999

Neutralizing capacity of antisera raised in horses and rabbits against Crotalus durissus terrificus (South American rattlesnake) venom and its main toxin, crotoxin.

Crotalus durissus terrificus (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. We have investigated the ability of commercial equine antivenom and antivenoms raised in rabbits against C. d. terrificus venom and crotoxin to neutralize the physiological and morphological changes induced by this venom and crotoxin in electrically-stimulated phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations of mice. The time required to produce 50% neuromuscular blockade in the PND and EDL preparations was, respectively, 103+/-9 and 59+/-6 min for C. d. terrificus venom (10 microg/ml) and 75+/-9 and 110+/-7 min for crotoxin (10 microg/ml). The antivenoms dose-dependently inhibited this neuromuscular activity of the venom and crotoxin. At a venom:antivenom ratio of 1:3, the rabbit antivenoms were as effective as the commercial equine antivenom. The creatine kinase (CK) concentrations in the organ bath containing EDL muscle were 290 and 1020 U/l following a 120 min exposure to C. d. terrificus venom and crotoxin, respectively. All of the antivenoms neutralized the release of CK by crotoxin, but were ineffective against C. d. terrificus venom. Histological analysis of the two preparations showed that rabbit anticrotoxin antivenom protected against the myotoxic action of C. d. terrificus venom and crotoxin better than the other antivenoms. We conclude that antisera raised in rabbits are better than equine antiserum in neutralizing the neurotoxic and myotoxic activities of C. d. terrificus venom and crotoxin.

Animals↗