title: An improved procedure for the isolation and purification of nicotinic acetylcholine receptor from Torpedo fuscomaculata electric organ.
abstract: The nicotinic acetylcholinergic receptor has been isolated and purified from extracts of the electric organ of the fish Torpedo fuscomaculata. The isolation procedure involves (a) a series of purification steps including preparation of membrane fragments, extraction of receptors with non-ionic detergents and chromatofocusing; (b) a novel fluorimetric titration assay. The purified receptor is isolated following a 9-fold purification with an overall yield of 12% and a specific activity of 4027 nM.g-1. Gel electrophoresis in the presence of sodium dodecylsulphate produced only one major band with molecular weight of 44,600 associated with the alpha-subunit. A comparison is made with other established procedures. Affinity chromatography on cobratoxin CNBr-Sepharose CL4B produced a 6.8-fold purification, 5% yield and 2900 nM.g-1 specific activity, while in ion-exchange chromatography on DEAE Sepharose 6B gave a 4.7-fold purification, 3% yield and specific activity of 1988 nM.g-1.
authors.affiliation: Department of Chemistry and Biochemistry, Rhodes University, Grahamstown, Republic of South Africa.
authors.forename: E A
authors.initials: EA
authors.lastname: Kapp
authors.forename: C G
authors.initials: CG
authors.lastname: Whiteley
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004557:Electric Organ / Q000032:analysis*; D008970:Molecular Weight; D011978:Receptors, Nicotinic / Q000302:isolation & purification*; D014101:Torpedo
chemical_list: D011978:Receptors, Nicotinic