Betz_1982_J.Biol.Chem_257_11390

Polypeptides involved in the binding of the nicotinic acetylcholine receptor ligand alpha-bungarotoxin (Mr = 8,000) to neuronal membranes were identified by three independent methods: (i) 125I-alpha-bungarotoxin bound to membrane fractions or to monolayer cultures of chick retina was cross-linked to its binding site by using glutaraldehyde, or the photoactivatable bifunctional reagent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Electrophoretic analysis of the cross-linked membrane proteins revealed 125I-alpha-bungarotoxin-polypeptide adducts of apparent Mr = 63,000, 43,000, and 33,000. (ii) Affinity purification of the alpha-bungarotoxin binding protein from detergent extracts of [35S]methionine-labeled retina cultures identified one major polypeptide with an Mr = 57,000. (iii) Indirect immunoprecipitation from detergent extracts of [35S]methionine-labeled rat pheochromocytoma cells (PC 12) gave evidence for a specific co-precipitation of alpha-bungarotoxin with three polypeptides (Mr = 57,000, 34,000, and 25,000). The data suggest that polypeptides of Mr - 57,000, 35,000, and 25,000 (+/- 3,000) are located at or close to the alpha-bungarotoxin binding domain of the putative neuronal nicotinic acetylcholine receptor.