Molecular Pharmacology, Vol 10, 1-14, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

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

I. Binding of the Tritiated -Neurotoxin in the Absence of Effector

¹ Neurobiologie Moléculaire, Institut Pasteur, Paris XV^e, France

Binding of a tritiated -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 -toxin behaved exactly like the native, unlabelled compound. The number of [³H]-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]-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]-toxin-membrane complex in the presence of an excess of unlabelled -toxin is about 60 hr. The equilibrium dissociation constant, estimated from the kinetic data, is 20 pM.

Note:
ACKNOWLEDGMENTS We thank Professor P. Boquet for purification and a generous gift of pure -toxin; Drs. A. Menez J. L. Morgat and P. Fromageot for its tritiation; Professor P. G. Waser for the gift of muscarone; and the Laboratoire Roger Bellon for the gift of dimethisoquin and prilocaine. We thank Drs. R. L. Baldwin, H. Buc, J. B. Cohen, G. L. Hazelbauer, H. Lester, J. C. Meunier, R. W. Olsen, and R. Sealock for helpful criticism and suggestions and aid in the preparation of the manuscript. We thank Dr. J. Patrick for the privileged communication of a manuscript in publication.

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