THE BINDING INTERACTION BETWEEN α‐LATROTOXIN FROM BLACK WIDOW SPIDER VENOM AND A DOG CEREBRAL CORTEX SYNAPTOSOMAL MEMBRANE PREPARATION

Abstract

Abstract—The major toxin of black widow spider venom, α‐latrotoxin, can be iodinated with 125I with hardly any loss in biological activity. The radioactive toxin could bind specifically to a dog cerebral cortex synaptosomal membrane preparation but not to a dog liver plasma membrane preparation. The bound protein could be recovered from the neuronal membrane preparation in an unchanged form. Non‐specific binding was only 6–10% of the total binding. The protein nature of the presumed receptor was indicated by the complete inhibition of the binding by either heating the membrane preparation at 70°C or treating the membrane with trypsin. Pre‐incubation with 2%β‐mercaptoethanol also completely inhibited the binding, while 70% inhibition was observed after pre‐treatment with 10m M‐EDTA or EGTA. From plots of the equilibrium binding data, it could be ascertained that the binding is non‐cooperative, with an apparent equilibrium dissociation constant, K1, of 1.0 nM. Kinetic data gave an apparent association rate constant of 8.2 × 105 M−1 s−1. Dissociation followed a biphasic exponential with rate constants of 1.4 × 10−3 and 5.2 × 10−5s−1 corresponding to half‐lives of 8.2 min and 3.7 h. Possible schemes for the binding interaction were proposed. Based on the present results and on previous results which indicated that α‐latrotoxin causes the release of all neurotransmitters and a depletion of the synaptic vesicle population in vertebrate synapses, a hypothetical mechanism of the action for the toxin was proposed, involving the binding of the toxin to a membrane protein receptor which interacts with filamentous proteins linking the synaptic vesicles to the axolemma.