Purification of a Cholinergic Receptor Isolated from Housefly Heads

Studies with aqueous extracts isolated from insect central nervous tissue such as housefly heads have shown that the cholinergic receptor, as judged by the ability to bind in a reversible manner a spectrum of cholinergic ligands, appears to be concentrated in a lOO000g supernatant fraction (O'Brien et al., 1972). The present paper reports the localization in a similar housefly head fraction (an 80000g supernatant) of a receptor-like material that binds with high affinity a number of cholinergic ligands such as acetylcholine, nicotine, atropine and decamethonium. We also describe further studies on the characterization and purification of the cholinergic receptor in aqueous extracts of housefly heads. In a preliminary attempt to clarify the nature of the housefly cholinergic receptor we have studied the ability of a variety of drugs, mainly of a nicotinic or muscarinic nature, to compete with radioactively labelled decamethonium for binding sites present in the 80000g supernatant fraction. Binding studies were performed with extracts that had been reconstituted in phosphate-free Ringer solution (Changeaux et a[., 1971) from the material obtained by the freeze-drying of the 80000g supernatant derived by the differential centrifugation of aqueous homogenates of housefly heads. The acetylcholinesterase(EC3.1.1.7)present insuchextracts was inhibitedasaroutine byapreincubation step with 0.lpM-paraoxon for lOmin at 25°C to prevent ligand binding to the catalytic site of the esterase. Reactionmixtures in Ringer solutioncontaining 1.8mgof protein in a volume of 0.5ml were incubated at 25°C for lOmin with a concentration range of the competing ligands before the addition of 1 ,UM [Me-3H]decamethonium. After a further incubation period of lOmin at 25"C, samples were analysed for bound decamethonium by using an ultrafiltration assay (Paulus, 1969). Table 1 shows the effect of the ligands on