Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.
A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.
D000468:Alkalies; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids; D004591:Electrophoresis, Polyacrylamide Gel; D005399:Fishes / Q000378:metabolism*; D007463:Iodobenzoates; D008094:Lithium; D008854:Microscopy, Electron; D011950:Receptors, Cholinergic / Q000302:isolation & purification*; D011978:Receptors, Nicotinic / Q000302:isolation & purification*; D012459:Salicylates; D013570:Synaptic Membranes / Q000032:analysis / Q000648:ultrastructure
D000468:Alkalies; D002793:Cholic Acids; D007463:Iodobenzoates; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D012459:Salicylates; C020891:3,5-diiodosalicylic acid; D008094:Lithium