Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins

Abstract

A rapid method for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from T. 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-iodosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the 4 polypeptides (MW 40,000, 50,000, 60,000 and 65,000) 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.