Acetylcholine receptor from mammalian skeletal muscle. Oligomeric forms and their subunit structures.
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 alpha-neurotoxin and lectin affinity gels, and shown to bind specifically 10--11.5 mumoles [2,3-3H]propionyl-alpha-bungarotoxin/g protein. The association rate constant (3 x 10(5) M-1 s-1 at 25 degrees 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 43,000 was detected in the pure protein. Likewise, both 9-S and 4-S oligomers isolated in a pure state at high yield (approximately equal to 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 43,000) 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.
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D002415:Cats; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D011950:Receptors, Cholinergic; D000109:Acetylcholine