The Acetylcholine Receptor: I. PURIFICATION AND CHARACTERIZATION OF A MACROMOLECULE ISOLATED FROM ELECTROPHORUS ELECTRICUS

Abstract

Abstract The nicotinic acetylcholine receptor has been isolated from extracts of the electric organ of the eel, Electrophorus electricus. The isolation procedure involves the following elements: (a) a rapid and reproducible assay that is based on the ability of receptor to bind cobra-[3H] neurotoxin and allows complex formation to be measured using DEAE-cellulose paper discs; (b) a series of purification steps including preparation of membrane fragments, extraction of receptors with nonionic detergents, followed by gel permeation, affinity and ion exchange chromatography. The purified receptor is isolated following 8250-fold purification with an over-all yield of approximately 5%. The receptor is a large protein that is free of ultraviolet absorbing prosthetic groups, and migrates as a single band in polyacrylamide gel electrophoresis both when native and denatured. The amino acid analysis distinguishes this protein from acetylcholinesterase. The best preparations bind 1 mole of neurotoxin per 90,000 g of protein, and the ability to form complexes is sensitive to heat and proteolysis. The molecular weight and subunit structure remain to be determined. The reaction between receptor and cobra neurotoxin proceeds toward the equilibrium state slowly. The apparent affinity of receptor for toxin increases with longer times of incubation. The toxin receptor equilibrium changes as a function of temperature, and this change is entirely due to changes in the rate of association, the dissociation rate remaining temperature independent. The kinetic and thermodynamic parameters at 25° are: k1 = 1.35 x 105 mole-1 s-1, k-1 = 1.52 x 10-4 s -1, Kd = 1.13 x 10-9 m, ΔH0 = 15.3 Cal per mole, ΔG0 = -12.2 Cal per mole, ΔS0 = +92 e.u. The toxin-receptor equilibrium and the DEAE-filter paper assay provide a convenient method for studying the interaction of receptor with cholinergic ligands.