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

Purified ol-bungarotoxin was prepared from the venom of Bungarus mulficincfus 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 ar-bungarotoxin was acetylated with (3H]acetic anhydride, and the [“Hlacetylated, purified o(bungarotoxin was called [3H]acetyl-a-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 10e5 g per ml of 13H]acetylcr-bungarotoxin caused a complete block of the indirectly stimulated rat hemidiaphragm in vifro, and this action was quantitatively and qualitatively similar to that of the unacetylated a-bungarotoxin. The [zH]acetyl-ar-bungarotoxin had an isoelectric point of pH 9.3. [aH]Acetyl-oc-bungarotoxin prepared in this manner had a specific activity of 6.36 Ci per mmole. Binding of [3H]acetyl-cY-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-oL-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 (10e2 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-cr-bungarotoxin could be bound. At a [3H]acetyl-a-bungarotoxin concentration of 0.875 PM, 1 pM d-tubocurarine inhibited binding 52 % ; 1 PM acetylcholine, 30% ; 1 pM choline, 0% ; 1 pM carbachol, 38 % ; 1 PM atropine, 0 % ; 1 PM gallamine, 33 % ; 1 m decamethonium, 44%; 1 pM hexamethonium, 16%; 1 PM propanpheline, 0% ; and 1 pM serotonin, 0%. A Scatchard plot indicated that two binding sites were present in the binding molecules with dissociation constants of 6.61 x 10ms M and 2.41 X lo-’ M. Calculation of binding site concentrations give Br of 12 mnoles per g of cerebral cortex wet weight and Ba of 26 mnoles per g of cerebral cortex wet