STUDIES OF REVERSIBLE AND IRREVERSIBLE INTERACTIONS OF AN ALKYLATING AGONIST WITH TORPEDO-CALIFORNICA ACETYLCHOLINE-RECEPTOR IN MEMBRANE-BOUND AND PURIFIED STATES

The interaction of a cholinergic depolarizing agent, bromoacetylcholine, with acetylcholine receptor (AcChR) enriched membrane fragments and Tritonsolubilized, purified AcChR from Torpedo californica has been studied. The reagent bound to membrane-bound AcChR reversibly with an apparent dissociation constant of 16 ± 1 nM at equilibrium. This 600-fold higher affinity for the receptor than found from physiological studies [Kact ≅10 μM; Karlin, A. (1973) Fed. Proc., Fed. Am. Soc. Exp. Biol. 32, 1847-1853] can be attributed to a ligand-induced affinity change of the membrane-bound receptor upon preincubation with bromoacetylcholine. At equilibrium [3H] bromoacetylcholine, like acetylcholine, bound to half the number of α -bungarotoxin sites present in the preparation without apparent positive cooperativity, and this binding was competitively inhibited by acetylcholine. In the presence of dithiothreitol, [3H]-bromoacetylcholine irreversibly alkylated both membrane-bound and solubilized, purified acetylcholine receptor, with a stoichiometry identical with that for reversible binding. NaDodSO4-polyacrylamide gel electrophoresis of the labeled acetylcholine receptor showed that only the 40000-dalton subunit contained the label. From these results it is concluded that the 40 000-dalton subunit represents a major component of the agonist binding site of the receptor. © 1979, American Chemical Society. All rights reserved.