Molecular Pharmacology, Vol 7, 538-553, Copyright © 1971 by the American Society for Pharmacology and Experimental Therapeutics

Studies on the Cholinergic Receptor Protein of Electrophorus Electricus

I. An Assay in Vitro for the Cholinergic Receptor Site and Solubilization of the Receptor Protein from Electric Tissue

¹ Département de Biologie Moléculaire, Institut Pasteur, Paris, France

The binding of ¹⁴C-decamethonium to a preparation of the electric organ of Electrophorus electricus has been measured by a method of rapid equilibriunm dialysis. At the ionic strength of Ringer's physiological solution little nonspecific binding of decamethonium occurs. Deoxycholate extraction of membrane fragments yields a preparation which contains two classes of specific decamethonium-binding sites. From one, the ligand is displaced reversibly by d-tubocurarine, gallamine triethiodide (at concentrations lower than 10^-5 M), carbamylcholine, and phenyltrimethylammonium, and irreversibly by two snake venom toxins, -bungarotoxin and Naja nigricollis -toxin. This class of site is considered to belong to the cholinergic receptor site. From the other, decamethonium is only displaced by carbamylcholine and phenyltrimethylammonium. This second class of site is identified as the catalytic site of acetylcholinesterase. The "intrinsic" binding constants of a variety of cholinergic agents for these two classes of sites are compared with their "apparent" values estimated in vivo on the isolated electroplax and in vitro on excitable membrane fragments. Some agreement exists between the two sets of data. The macromolecule possessing the cholinergic receptor site has a molecular weight larger than 50,000 daltons, is thermolabile, and is digested by Pronase. It is a protein that is easily separated from acetylcholinesterase by selective adsorption on Sepharose granules to which N. nigricollis -toxin has been coupled.

Note:
ACKNOWLEDGMENTS The authors thank Drs. R. Olsen and J. Patrick for helpful comments and criticisms, Dr. C. Y. Lee for the gift of a sample of pure -bungarotoxin, and Dr. P. Boquet for the supply of important quantities of pure -toxin from Naja nigricollis.

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