Acetylcholine Receptor from Mammalian Skeletal Muscle

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Abstract
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 [2,3-3H]propionyl-α-bungarotoxin/g protein. The association rate constant (3 × 105 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 Mr 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[3H]acetylcholine. As in the case of the pure receptor from denervated muscle, the same size polypeptide (Mr 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.