Acetylcholine Receptor from Mammalian Skeletal Muscle

1 The acetylcholine receptor of cat denervated skeletal muscle was solubilised with Triton X‐100 in the presence of protease inhibitors and was shown to have a sedimentation coefficient of about 9 S. This oligomer can be converted to a smaller, active 4‐S species. 2 This 9‐S glycoprotein was purified to homogeneity (showing pI = 5.0) by improved biospecific chromatography on a‐neurotoxin and lectin affinity gels, and shown to bind specifically 10–11.5 μmol [2,3‐ 3 H]propionyl‐α‐bungarotoxin/g protein. The association rate constant (3 × 10 5 M −1 s −1 at 25°C) for this reaction was similar to that observed with membrane‐bound or unpurified receptor; affinity constants for nicotinic ligands were also similar in all these cases. 3 By a variety of techniques, a major polypeptide of M r about 43000 was detected in the pure protein. Likewise, both 9‐S and 4‐S oligomers isolated it a pure state at high yield (∼ 80%) by a novel technique using anti‐toxin immunoglobulin, contained the same size of subunit. 4 Sub‐synaptic and extra‐synaptic forms of the receptor were alkylated specifically in the membrane‐bound state with the affinity reagent bromo[ 3 H]acetylcholine. As in the case of the pure receptor from denervated muscle, the same size polypeptide ( M r 43000) was labelled. This was true, also, for both the 9‐S and the 4‐S oligomer of the denervated muscle receptor. 5 Proposed oligomeric structures of acetylcholine receptors containing single and multiple‐size subunits are discussed.