Acetylcholine receptor. I. Identification and biochemical characteristics of a cholinergic receptor of guinea pig cerebral cortex.

Abstract Purified α-bungarotoxin was prepared from the venom of Bungarus multicinctus by carboxymethyl Sephadex C-50 chromatography and Sephadex G-25 chromatography. The material was found to be a pure polypeptide of molecular weight 8000. The purified α-bungarotoxin was acetylated with [3H]acetic anhydride, and the [3H]acetylated, purified α-bungarotoxin was called [3H]acetyl-α-bungarotoxin and was utilized to characterize and isolate an acetylcholine receptor from guinea pig cerebral cortex. With an isolated rat phrenic nerve diaphragm preparation, 1 x 10-5 g per ml of [3H]acetyl-α-bungarotoxin caused a complete block of the indirectly stimulated rat hemidiaphragm in vitro, and this action was quantitatively and qualitatively similar to that of the unacetylated α-bungarotoxin. The [3H]acetyl-α-bungarotoxin had an isoelectric point of pH 9.3. [3H]Acetyl-α-bungarotoxin prepared in this manner had a specific activity of 6.36 Ci per mmole. Binding of [3H]acetyl-α-bungarotoxin was measured in a 0.1% Triton X-100, 0.1 m Tris (pH 7.6) homogenate of guinea pig cerebral cortex with an ultrafiltration cell apparatus. Binding of [3H]acetyl-α-bungarotoxin to the homogenate was linear with an added homogenate protein, was reversible, was saturable, showed optimum binding at pH 7.1 to pH 7.7, and was not inhibited by high concentrations (10-2 m) of physostigmine. With excess 0.1% Triton X-100, 0.1 m Tris (pH 7.6) homogenate of guinea pig cerebral cortex, essentially 100% of the available [3H]acetyl-α-bungarotoxin could be bound. At a [3H]acetyl-α-bungarotoxin concentration of 0.875 µm, 1 µm d-tubocurarine inhibited binding 52%; 1 µm acetylcholine, 30%; 1 µm choline, 0%; 1 µm carbachol, 38%; 1 µm atropine, 0%; 1 mum gallamine, 33%; 1 µm decamethonium, 44%; 1 µm hexamethonium, 16%; 1 µm propanpheline, 0%; and 1 µm serotonin, 0%. A Scatchard plot indicated that two binding sites were present in the binding molecules with dissociation constants of 6.61 x 10-8 m and 2.41 x 10-7 m. Calculation of binding site concentrations give B1 of 12 nmoles per g of cerebral cortex wet weight and B2 of 26 nmoles per g of cerebral cortex wet weight. Treatment of the homogenate with proteolytic enzymes decreased binding; deoxyribonuclease, ribonuclease, lipase, phospholipase C, and neuraminidase were without effect. A receptor-[3H]acetyl-α-bungarotoxin complex was isolated and purified by centrifugation and Sephadex G-100, G-150, and G-200 column chromatography. The purified [3H]acetyl-α-bungarotoxin-receptor complex was homogeneous by polyacrylamide gel electrophoresis, had an isoelectric point of pH 4.8, and has a molecular weight of 94,000. The purified [3H]acetyl-α-bungarotoxin-receptor complex had the properties of protein, indicating that the receptor is probably a protein. Fractionation of the cerebral cortex showed an enrichment of the receptor in the synaptosomal fraction. Subsynaptosomal fractionation showed the synaptosome plasma membrane to contain the highest concentration of receptors. The original cerebral cortex homogenate bound 87.5 ± 7 pmoles of [3H]acetyl-α-bungarotoxin per mg of protein, which, assuming one molecule of [3H]acetyl-α-bungarotoxin per receptor molecule, means that a 3 g (wet weight) guinea pig cerebral cortex contains 52.5 nmoles of receptor or 420 µg of receptor protein. The purified [3H]acetyl-α-bungarotoxin-receptor complex contained 10.75 nmoles of [3H]acetyl-α-bungarotoxin per mg of protein or 86 ± 4 µg of [3H]acetyl-α-bungarotoxin per mg of protein.