Binding of Naja nigricollis [³H]α-Toxin to Membrane Fragments from Electrophorus and Torpedo Electric Organs

Abstract

Binding of a tritiated α-neurotoxin from Naja nigricollis to membrane fragments purified from electric tissues of Electrophorus electricus and Torpedo marmorata was measured by ultrafiltration on Millipore filters. Isotopic dilution and pharmacological experiments showed that the tritiated α-toxin behaved exactly like the native, unlabelled compound. The number of [³H]α-toxin binding sites on membrane fragments is about 10 nmoles/g of protein for Electrophorus and 1000 nmoles/g for Torpedo. The kinetics of association of [³H]α-toxin with the membrane is compatible with a bimolecular mechanism of binding to a homogeneous class of sites. The second-order rate constant of association is 2.5 x 10⁷ M^-1 min^-1 at 20° in Ringer’s solution. It decreases with increasing ionic strength and sucrose concentration. The half-time for dissociation of the [³H]α-toxin-membrane complex in the presence of an excess of unlabelled α-toxin is about 60 hr. The equilibrium dissociation constant, estimated from the kinetic data, is 20 pM.