Purification and properties of the acetylcholine receptor protein from Narke japonica.

Abstract

The electric organ of Narke japonica was found to contain 0.4–1.11 nmol of [3H]toxin α (Naja nigricollis) binding sites per gram tissue. The nicotinic acetylcholine receptor protein from the electric organ of the Japanese electric ray was purified about 80-fold with an overall yield of 19% from the whole homogenate of the electric organs. The purification procedure included the following procedures: (1) preparation of membrane fragments from the electric organs and solubilization of the receptor from them with a nonionic detergent; (2) DEAE-cellulose chromatography of the solubilized receptor; and (3) affinity chromatography of the DEAE-cellulose fraction using a weak snake neurotoxin, Laticauda semifasciata III (Laticauda semifasciata), as an affinity ligand. The reversibility of the receptor-toxin binding permitted elution of the receptor under mild conditions. The specific toxin binding activity of the final preparation was 2.17 nmol/mg protein. The preparation showed no acetylcholinesterase activity. The preparation gave single coinciding major bands of protein and toxin binding activity upon polyacrylamide gel disc electrophoresis. Upon gel electrophoresis in the presence of sodium dodecyl sulfate, two major bands corresponding to molecular weights of 33, 000 and 43, 000, and two minor bands of 38, 000 and 51,000 were detected. The toxin binding by purified receptor protein was inhibited to various extents by cholinergic agonists and antagonists. The purified receptor protein was found to contain carbohydrate (18%).