The role of crotoxin subunits in tropical rattlesnake neurotoxic action.

The major toxin (crotoxin) of Crotalus durissus terrificus (neotropical rattlesnake) is known to be a reversible non-covalently associated complex consisting of an acidic and basic subunit. On separation biological activity is found only with the basic subunit, yet, although void of detectable biological activity, the acidic subunit is essential for the full neurotoxic activity of the complex. Recent evidence suggests that crotoxin A serves as a 'chaperone' to enhance the specificity of crotoxin B and, upon binding, crotoxin A is released to the medium. This study was designed to test this hypothesis. Dimethyl suberimidate, a bifunctional cross-linking agent, was used to irreversibly bind the two subunits. Disc electrophoresis, ion-exchange chromatography, molecular sieve chromatography, capillary isotachophoresis and isoelectric precipitation confirm the existence of an inter-subunit covalently cross-linked complex. The conversion of a dissociable complex to a non-dissociable complex abolished neurotoxicity. Although neurotoxicity was lost, phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4), which is found associated with many presynaptic neurotoxins, was unaffected. The data in this paper add credence to the 'chaperone' concept of crotoxin A and the importance of the reversible nature of the complex for full expression of neurotoxicity.

Biochemical characterization of the lizard toxin gilatoxin.

The Gila monster (genus Heloderma) is the only known lizard to produce and inject a venomous secretion. Little is known about the venom from these lizards, and none of the toxins have been isolated until this time. This paper reports the isolation and characterization of a major lethal toxin (gilatoxin) from the venoms of Heloderma suspectum and Heloderma horridum. Gilatoxins from both species were similar in amino acid composition, electrophoretic mobility, pI, and immunological reactivity. They are acidic proteins possessing molecular weights of 35 000-37 500 and isoelectric points of 4.25 and consist of a single polypeptide chain. Neither is antigenically related to the venoms of snakes. The toxins are devoid of phospholipase A2 activity and proteolytic, hemorrhagic, and hemolytic activities, with lethality being the only biological activity detectably expressed. The toxins appear to be unique and distinct from those of other venomous animals.

Biological roles of the two components of crotoxin.

Crotoxin, the Brazilian rattlesnake neurotoxin, generally behaves as a homogeneous protein; however, it is a molecular complex of an acidic and a basic protein. These can be separated after alkylation or acylation of the amino groups, or on carboxymethyl cellulose at pH 4, or on DEAE-cellulose in 6 M urea. The two proteins differ greatly in composition, but one or both may exist in the form of closely related variants. Their molecular weights appear to be about 8400 and 13,000. The acidic protein lacks the hemolytic and neurotoxic activity of crotoxin and the basic protein shows only the high, indirect hemolytic activity; a mixture of the two components shows the high neurotoxicity of crotoxin.

Search for relationships among the hemolytic, phospholipolytic, and neurotoxic activities of snake venoms.

Several snake venom neurotoxins are larger and more complex than the well-studied group of postsynaptic toxins exemplified by alpha-bungarotoxin. Several of these, exemplified by beta-bungarotoxin, show phospholipase A2 activity (phosphatide 2-acylhydrolase, EC 3.1.1.4) when tested in the presence of detergents. The high hemolytic activity of crotoxin, the neurotoxin of Crotalus durissus terrificus, in the presence of lecithin has been attributed to this activity. The phospholipase A2 activity of several snake venom proteins has now been compared under the physiological conditions of the hemolysis tests. It appears that only the basic component of crotoxin, B, is enzymatically active, and that its activity is not inhibited by component A under these conditions, or in the presence of deoxycholate. Phosphatidylserine is found to be digested more readily than egg white phosphatidylcholine; and also causes hemolysis in conjunction with much lower levels of crotoxin. In neither case is calcium required or stimulating. Phospholipase from Crotalus adamanteus, which is not neurotoxic, digests phosphatidylcholine more rapidly than does crotoxin, but phosphatidylserine more slowly; yet it is slightly less active than crotoxin in the hemolysis test with phosphatidylcholine, and much less with phosphatidylserine. The digestion of several phospholipids by either enzyme fails to release the expected protons in the absence of detergents at 37 degrees .beta-Bungarotoxin, highly neurotoxic, has negligible phospholipase A2 activity in the absence of detergents, and is almost nonhemolytic in conjunction with all phospholipids tested.Binding studies with (125)I-labeled compounds show that rabbit erythrocytes and ghosts have much greater affinity for crotoxin than for beta-bungarotoxin and do not bind Crotalus adamanteus phospholipase. The crotoxin complex is split in the course of binding, with only component B, the hemolytic component, becoming bound. It appears that the role of component A may be to diminish the nonspecific binding tendency of component B. Our data appear to be consistent with the concepts that affinity to membranes, particularly to specific sites on synaptic membranes, is the critical requirement for beta type neurotoxicity, and that this property, at least in some instances, has evolved from phospholipase A2 enzymes, but does not necessarily require retention and expression of enzymatic activity.