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Reconstitution of a functional acetylcholine receptor. Polypeptide chains, ultrastructure, and binding sites for
Abstract:
The 'reconstitution cycle' is composed of the following sequence of operations. Highly purified receptor-rich membranes prepared from Torpedo marmorata electric organ are exposed to pH 11 to remove the 43,000-Mr protein and dispersed into solution by sodium cholate under conditions where more than 85% of the receptor protein is in its 9-S form. Elimination of the detergent by filtration on a Sephadex column (or dialysis) yields a 'reconstituted receptor' fraction, under conditions which conserve part of the endogenous lipids, or 'reconstituted vesicles' in the presence of an excess of exogenous lipids. The polypeptide composition of these fractions was analysed by sodium dodecylsulfate gel electrophoresis. Conditions are defined for quantitative measurements of the various polypeptide chains. The 40,000-Mr chain, which is labelled by the affinity reagent 4-(N-maleimido)phenyl [3H]trimethylammonium and therefore carries the acetylcholine receptor site, is the dominant polypeptide in the alkaline-treated membranes and the reconstituted acetylcholine receptor. Electron microscopy discloses that many of the alkaline-treated membranes no longer form closed vesicles and do not show the transverse asymmetry of the native membranes observed after tannic acid fixation. In the reconstituted receptor fractions, the receptor molecules reaggregate into discs and may be exposed on both faces of the discs. In the reconstituted vesicles, receptor rosettes are integrated to the lipid vesicles. With native membranes, the radioactive local anesthetic [3H]trimethisoquin binds to three classes of sites: non-specific, low-affinity and high-affinity. Carbamylcholine causes an increase in the number of high-affinity sites up to approximately 0.7 times the number of alpha-125I-bungarotoxin sites. This ratio, the three classes of binding sites, and their regulation by carbamylcholine are conserved through the reconstitution cycle.