A y-Aminobutyric Acid/Benzodiazepine Receptor Cerebral Cortex

The y-aminobutyric acid/benzodiazepine receptor from bovine cerebral cortex was solubilized with sodium deoxycholate and purified by affinity chromatography on benzodiazepine-agarose and ion exchange chromatography. The benzodiazepine binding protein was enriched 1800-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of two major bands of M, = 57,000 and 53,000. [3H]Flunitrazepam, after UV irradiation, was incorporated irreversibly into both bands of the isolated protein. A high affinity binding site for y-aminobutyric acid was co-purified with the benzodiazepine binding site and the two sites were shown to reside on the same physical structure. The dissociation constants were 10 2 4 nM for [3H] flunitrazepam and 12 2 3 nM for the y-aminobutyric acid agonist ~3H]muscimol. The maximum specific activity for [3H]muscimol binding was 4.3 nmollmg of protein. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was between 3 and 4. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 7.3 0.5 nm and a sedimentation .coefficient of 11.1 f 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds*to the receptor specificity found in membranes and crude soluble extracts.