Investigations on the mechanism of action of crotoxin.
Crotoxin, the major toxic protein of Crotalus durissus terrificus, is composed of a basic phospholipase, component-B, and of an acidic subunit, component-A. The crotoxin complex is insensitive to an active site directed reagent, p-bromophenacyl bromide, while its isolated enzymatic component-B is rapidly and irreversibly inactivated. We observed that crotoxin possesses an intrinsic phospholipase A2 activity on monodispersed substrates, indicating that the active site of component-B is not masked by component-A in the complex. The inactivation of component-B by p-bromophenacyl bromide follows pseudo-first order kinetics, with a rate constant proportional to the concentration of phospholipase, as expected for a reaction of second order with respect to the protein. On the basis of a detailed kinetic analysis of this reaction, and of physico-chemical studies of component-B in various experimental conditions, we propose that (1) an equilibrium exists between reactive dimers of component-B and preponderant but non-reactive monomer; (2) component-A protects component-B against inactivation by p-bromophenacyl bromide by preventing the formation of reactive dimers. When comparing the reactivity of component-B with that of other phospholipases, we observed that the enzymes which have not been shown to produce dimers all react with p-bromophenacyl bromide with similar low rates of reaction, while phospholipases which have been reported to form dimers react much more rapidly.
D000098:Acetophenones / Q000494:pharmacology; D000478:Alkylation; D000818:Animals; D001665:Binding Sites; D003435:Crotalid Venoms / Q000494:pharmacology*; D003439:Crotoxin / Q000378:metabolism / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D009498:Neurotoxins / Q000494:pharmacology*; D010740:Phospholipases / Q000378:metabolism
D000098:Acetophenones; D003435:Crotalid Venoms; D009498:Neurotoxins; D003439:Crotoxin; D010740:Phospholipases; C006821:4-bromophenacyl bromide