1. The affinity alkylation reaction of the cholinergic, depolarising ligand, bromoacetylcholine with reduced acetylcholine receptor in the membrane fragments of Torpedo marmorata and in Triton-solubilised receptor from cat denervated muscle has been studied.2. Brief pretreatment with 100 pM bromoacetylcholine abolishes all [3H]a-neurotoxin binding in both cases.3. In the receptor from each of these sources, the number of sites of specific a-neurotoxin binding is exactly equal to the number of sites that can be specifically alkylated by br~mol [~H]acetylcholine, at saturation of either ligand.4. The concentration-dependence of specific br~mo[~H]acetylcholine binding is found to be biphasic. A first phase can be clearly discerned in which one-half of the total specific ligand-binding sites are alkylated readily, and a second phase in which the remainder react at higher reagent concentrations. The same discrimination of two equal sets of ligand sites can be obtained by preblockade using low concentrations of unlabelled bromoacetylcholine followed by reaction with [3H]a-neurotoxin or bromo[3H]acetylcholine.5. In both phases, a single subunit of M , about 43000 is the sole site of specific alkylation in both Torpedo and muscle. The reasons for the appearance of two equal but distinct populations in the ligand binding sites in the receptors are discussed.A number of studies on the nicotinic acetylcholine receptor have endeavoured to quantify the number of binding sites for nicotinic ligands per molecule in this oligomeric protein [l -31. Measurements of the essentially irreversible binding of the specific antagonists, a-neurotoxins, to purified preparations of acetylcholine receptor, together with determination of their molecular weights, allow the number of toxin binding sites per oligomer to be estimated (for a review see [4]). However, the complicated pharmacology of the acetylcholine receptor complex [4], uncertainty in the exact receptor molecular weight, and readily reversible binding of quaternary ammonium effectors [5], are factors that have hindered the determination of the number of binding sites for Abbreviations. (cHxN)*C, dicyclohexylcarbodiimide; iPrZP-F, diisopropylfluorophosphate; T/L, stoichiometric ratio where T and L represent the total number of moles of toxin or ligand, respectively, that bind specifically to a given preparation of acetylcholine receptor.agonists. In practice, therefore, results have been related to a-toxin binding using a stoichiometric ratio TIL, where T and L represent the total number of moles of toxin or ligand, respectively, that bind specifically to a given preparation. Ligand binding in Torpedo has been investigated using the radiolabelled affinity reagent maleimido-benzyltrimethylammonium iodide [6], or more recently, bromoacetylcholine [7,8]. These reagents can specifically alkylate the acetylcholine receptor if a disulphide bond adjacent to the ligand binding site(s) has first been reduced [6,9] and studies on the Torpedo acetylcholine receptor have indicated a stoichio...
