Relationship between the Pharmacological Action of Crotoxin and Its Phospholipase Activity

Abstract

Crotoxin, the major neurotoxin from Crotalus durissus terrificus venom, blocks the postsynaptic response of Electrophorus electroplaque to bath‐applied carbamoylcholine. Crotoxin is composed of two non‐covalently linked subunits: an acidic non‐enzymatic component, A, and a basic phospholipase component, B. According to our previous studies, component A restricts the binding of component B to high‐affinity sites which occur in Torpedo membrane fragments in approximatively equivalent amount to the cholinergic receptor sites. We demonstrate here that the catalytic activity of component B is actually required for the postsynaptic blocking action of the toxin. Irreversible inactivation of component B was achieved by alkylation with p‐bromophenylacyl bromide of a histidine residue in the catalytic site. Although the native crotoxin complex was insensitive to the inhibitor, suggesting that the catalytic center is protected by component A, we obtained an enzymatically inactive crotoxin by recombination of the alkylated component B with component A. This modified crotoxin was devoid of enzymic activity, toxicity and pharmacological activity on the electroplaque. The phospholipase activity of component B may be reversibly inhibited by replacing Ca2+ with Sr2+ ions in the physiological solution. This allowed us to identify several steps in the action of the toxin: a quasi‐irreversible binding step which occurs either in the presence of Ca2+ or Sr2+ ions, and a catalytic step which requires Ca2+ ions and is responsible for the irreversible blockade of the electroplaque response to cholinergic agonists. Finally, we examined the effect of various toxic and non‐toxic phospholipases on the electroplaque response. The data exclude that the postsynaptic blockade results from indiscriminate hydrolysis of phospholipidb. They rather suggest that the pharmacological efficiency of crotoxin is due to the fact that component B exerts its catalytic activity at critical postsynaptic sites. These conclusions are in excellent agreement with the explanation of the synergistic effect of the two subunits which we derived from our binding data. The postsynaptic sensitive sites for crotoxin action might be closely related to the acetylcholine receptor sites. Copyright © 1982, Wiley Blackwell. All rights reserved