No abstract

Crotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus is a potent neurotoxin consisting of a weakly toxic phospholipase-A, 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 nonspecific 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-A, 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 &and V,, 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 (Kp25 nM) and dissociate rapidly (half-life about 1 min), whereas the more-toxic isofonns are more stable (Kp4.5 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 isofonns 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.The B neurotoxins from snake venoms are potent neurotoxic phospholipases A2 which block neuromuscular transmission by reducing the release of acetylcholine evoked by nerve stimulation (for reviews, see [l-31). Different classes of / 3 neurotoxins have been defined according to their quaternary structure. Some, such as notexin, agkistrodotoxin or ammodytoxin-A, are single-chain polypeptides homologous to non-toxic secreted phospholipases A,; others, such as crotoxin, taipoxin or textilotoxin, are non-covalent associations of several subunits with structures related to secreted phospholipases A,; finally, P-bungarotoxins consist of a phospholipase-A, subunit covalently associated with a Kunitztype trypsin inhibitor.Crotoxin from Crotalus durissus terrificus venom [41 has two non-identical subunits

Crotoxin, the major toxin of the venom of the South American rattlesnake, Crotalus durissus terrificus, is made of two subunits: component B, a basic and weakly toxic phospholipase A2, and component A, an acidic and nontoxic protein that enhances the lethal potency of component B. Crotoxin is a mixture of isoforms that results from the association of several isoforms of its two subunits. In the present investigation, we have purified four component A isoforms that, when associated with the same purified component B isoform, produced different crotoxin isoforms, all having the same specific enzymatic activity and the same lethal potency. We further determined by Edman degradation the polypeptide sequences of these four component A isoforms. They are made of three disulfide-linked polypeptide chains (alpha, beta, and gamma) that correspond to three different regions of a phospholipase A2 precursor. We observed that the polypeptide sequences of the various component A isoforms all agree with the sequence of an unique precursor. The differences between the isoforms result first by differences in the length of the various chains alpha and beta, indicating that component A isoforms are generated from the proteolytic cleavage of the component A precursor at very close sites, possibly by the combined actions of endopeptidases and exopeptidases, and second by the possible cyclization of the alpha-NH2 of the N-terminal glutamine residue of chains beta and gamma. These observations indicate that the component A isoforms are the consequence of different posttranslational events occurring on an unique precursor, rather than the expression of different genes.

An antivenom protein has been identified in the blood of the snake Crotulus durissus terriJicus and proved to act by specifically neutralizing crotoxin, the main lethal component of rattlesnake venoms. The aim of this study was to purify the crotoxin inhibitor from Crotulus serum (CICS), and to analyze its mechanism of action.CICS has been purified from blood serum of the Crotulus snake by gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephacel, and FPLC gel filtration on a Superose 12 column. It is an oligomeric glycoprotein of 130 kDa, made by the non-covalent association of 23 -25-kDa subunits. Two different subunit peptides were identified by SDSPAGE, however, their N-terminal sequences are identical. They are characterized by the absence of methionine residues and a high content of acidic, hydrophobic and cysteine residues.The neutralizing effect of purified CICS towards the neurotoxic effects of crotoxin has been demonstrated in vivo by lethality assays. CICS binds to the phospholipase subunit CB of crotoxin, but not to the acidic chaperon subunit CA; it efficiently inhibits the phospholipase activity of crotoxin and its isolated CB subunit and evokes the dissociation of the crotoxin complex.The molecular mechanism of the interaction between CICS and crotoxin seems to be very similar to that of crotoxin with its acceptor. It is, therefore, tempting to suggest that CICS acts physiologically as a false crotoxin acceptor that would retain the toxin in the vascular system, thus preventing its action on the neuromuscular system.