An improved procedure for the isolation and purification of nicotinic acetylcholine receptor from Torpedo fuscomaculata electric organ.
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.
Kapp
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EA
Department of Chemistry and Biochemistry, Rhodes University, Grahamstown, Republic of South Africa.
Whiteley
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CG
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
D011978:Receptors, Nicotinic