A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex.
The gamma-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 Mr = 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 gamma-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 +/- 4 nM for [3H] flunitrazepam and 12 +/- 3 nM for the gamma-aminobutyric acid agonist [3H]muscimol. The maximum specific activity for [3H] muscimol binding was 4.3 nmol/mg 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 +/- 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds to the receptor specificity found in membranes and crude soluble extracts.
D000818:Animals; D001569:Benzodiazepines / Q000378:metabolism*; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism*; D005445:Flunitrazepam / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009118:Muscimol / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D009118:Muscimol; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam