The purification and molecular characterisation of a putative nicotinic-muscarinic acetylcholine receptor from housefly heads

The purification and characterisation of a (³H)-decamethonium binding component which is a putative acetylcholine receptor with a mixed nicotinic-muscarinic pharmacology is described. The protein fraction obtained after differential centrifugation and gel permeation chromatography bound up to 1.8 μmoles of decamethonium/g protein and was shown to be low in acetylcholine esterase and other contaminating proteins. Preparative isoelectrofocusing of the purified putative receptor fraction produced two protein bands, with pI values of 4.7 and 4.9, which bound 0.65 and 0.1 μmoles of decamethonium/g protein respectively. SDS-polyacrylamide gel elctrophoresis of the purified putative receptor revealed the presence of two glycopeptides with molecular weights of 83,000 and 91,000. Polyacrylamide gel electrophoresis in the absence of SDS also revealed two glycopeptide bands. At least one of the polypeptides was shown to covalently bind the muscarinic antagonist (³H)-propylbenzilylcholine mustard. Cross-linkage of the solubilised protein with various cross-linking reagents produced a protein molecule with a molecular weight between 320,000 and 380,000. The putative acetylcholine receptor was shown to contain 3% by weight of neutral sugars (galactose, mannose and glucose) and between 1.9 and 2.4% by weight of glucosamine. Amino acid analysis of the purified protein indicates that it contains a high proportion of hydrophilic and polar residues and may therefore be a peripheral membrane protein.