Purification and properties of the cholinergic receptor protein from Electrophorus electricus electric tissue.

Abstract

The acetylcholine (nicotinic) receptor from the electric organ of Electrophorus electricus has been purified approx. 300-fold from Triton X-100 extracts of electroplax membrane fragments. The receptor protein was first adsorbed from crude extracts on an affinity column containing 1,3-bis(tri-ethylammonium ethoxy)-4-iodoacetamidobenzene diiodide, an analogue of flaxedil, linked to Sepharose 2B, then eluted with a solution of flaxedil and concentrated. The receptor protein was further purified by centrifugation on sucrose gradients containing 1 Triton X-100, 1% Emulphogene, or 0.5% sodium cholate. If desired, detergent could be removed after centrifugation in the presence of sodium cholate by gel filtration on Sephadex G-75. At all stages the receptor was assayed by incubation with α-[3H]toxin from Naja nigricollis followed by isolation of the toxin · receptor complex by filtration on Millipore filters at low detergent concentration. The best preparations bound 1 mol toxin per 150000 g protein. In the presence of non-denaturing detergents the preparations gave a single major band for both protein and activity upon polyacrylamide disc gel electrophoresis or sucrose gradient centrifugation. The receptor protein appears essentially homogeneous in the electron microscope after negative staining with uranyl acetate. Upon gel electrophoresis in the presence of dodecylsulphate, there were two major bands corresponding to molecular weights of 43000 and 48000. The ultraviolet-absorption spectrum and amino-acid composition of the purified receptor protein are typical of those of globular, water-soluble proteins in general and do not reflect the hydrophobic character of the macromolecule. Furthermore, the purified protein interacts with concanavalin A and other plant agglutinins indicating that it contains a carbohydrate moiety. The purified receptor protein carries a single class of binding sites for [3H]decamethonium (Kd= 0.02 μM) at ligand concentrations up to 1 μM and for acetylcholine (Kd= 0.06 μM) at up to 1 μM. No evidence for cooperativity in binding of cholinergic effectors to the receptor protein, either purified or in crude extracts, has been detected and all effectors tested inhibit the binding of [3H]decamethonium in a competitive manner. The affinity of agonists for the solubilized receptor protein is approx. 20-fold greater than for the membrane-bound protein.