Distinct protein components from Torpedo marmorata membranes carry the acetylcholine receptor site and the binding site for local anesthetics and histrionicotoxin.
Highly purified subsynaptic membrane fragments prepared from Torpedo marmorata electric organ (specific activity, greater than 4 mumol of Naja nigricollis alpha-[3H]toxin per mg of protein) exhibit, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, two major protein bands of apparent molecular weight 40,000 and 43,000, respectively. Dissolution of these membranes by the nondenaturing detergents Triton X-100 and Berol 043 followed by standard fractionation yielded (i) the 9S acetylcholine-receptor protein which still binds the alpha-[3H]toxin and after further purification yielded, in the presence of sodium dodecyl sulfate, the 40,000-dalton component, covalently labeled by the affinity reagent 4-(N-maleimido)phenyl[3H]trimethylammonium; only serine was found as the NH2-terminal amino acid of this protein; and (ii) a high molecular weight aggregate named 43,000 protein which was resolved in denaturing gels almost exclusively as the 43,000-dalton band, In the absence of detergents, the 43,000 protein binds compounds known to interact with the acetylcholine ionophore: a fluorescent local anesthetic quinacrine and histrionicotoxin (apparent dissociation constant, 7 +/- 1 X 10(-7) M). The regulation of quinacrine fluorescennce by carbamylcholine, observed in the intact membrane, no longer occurs with the isolated 43,000 component.
D000596:Amino Acids / Q000032:analysis; D000664:Amphibian Venoms / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004546:Elapid Venoms / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism*; D005399:Fishes; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010455:Peptides / Q000032:analysis; D011796:Quinacrine / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000032:analysis / Q000378:metabolism*
D000596:Amino Acids; D000664:Amphibian Venoms; D004546:Elapid Venoms; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D011950:Receptors, Cholinergic; D011796:Quinacrine
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