Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

Abstract

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin ( Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 m m benzoquinonium produces a protein that binds α-[ 125I]bungarotoxin but not [ 3H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [ 3H]acetylcholine, [ 3H]decamethonium, [ 3H]nicotine, [ 14C]dimethyl- d-tubocurarine, and α-[ 125I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing. The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity ( K D = 0.02 μM) and 5.1 nmoles a lower affinity ( K D = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μ m. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.