Stoichiometry of the Ligand-Binding Sites in the Acetylcholine-Receptor Oligomer from Muscle and from Electric Organ. Measurement by Affinity Alkylation with Bromoacetylcholine

Abstract

The affinity alkylation reaction of the cholinergic, depolarizing ligand, bromoacetylcholine with reduced acetylcholine receptor in the membrane fragments of Torpedo marmorata and in Triton-solubilized receptor from cat deneravated muscle was studied. Brief pretreatment with 100 .mu.M bromoacetylcholine abolishes all [3H].alpha.-neurotoxin binding in both cases. In the receptor from each of these sources, the number of sites of specific .alpha.-neurotoxin binding is exactly equal to the number of sites that are specifically alkylated by bromol[3H]acetylcholine, at saturation of either ligand. The concentration-dependence of specific bromo[3H]acetylcholine binding is biphasic. A 1st phase is clearly discerned in which 1/2 of the total specific ligand-binding sites are alkylated readily. In a 2nd phase, the remainder react at higher reagent concentrations. The same discrimination of 2 equal sets of ligand sites are obtained by preblockade using low concentrations of unlabeled bromoacetylcholine followed by reaction with [3H].alpha.-neurotoxin or bromo[3H]acetylcholine. In both phases, a single subunit of Mr about 43,000 is the sole site of specific alkylation in Torpedo and muscle. The reasons for the 2 equal but distinct populations in the ligand binding sites in the receptors are discussed.