Purification of ?-aminobutyric acid receptor, benzodiazepine receptor and C1 channel from bovine cerebral cortex by benzodiazepine affinity gel column chromatography.

Purification of solubilized ?-aminobutyric acid (GABA) and benzodiazepine receptors by Nonidet P-40 from the bovine cerebral cortex was employed by the affinity column chromatography using a benzodiazepine, 1012-S, as immobilized ligand. Although this benzodiazepine affinity gel retained all of the solubilized benzodiazepine receptors from synaptic membranes applied to the column, 25.6% of the GABA receptor binding activity was recovered in the run-through fraction. After the washing with NaCl, the biospecific elution with 1 mM 1012-S resulted the elution of 31.3% of GABA receptor binding activity applied to the column. The highest purification fold thus obtained was 3789 (specific activity: 0.39 nmol/mg protein). Successive application of 1-2 mM NaSCN also resulted further elution of GABA receptor. Scatchard analysis of [(3)H]muscimol binding to these fractions indicated that the purified GABA receptor had high and low affinity binding sites as detected in solubilized and synaptic membrane fractions. In addition, it was found that the B(max) values for both binding sites in the 1012-S-eluted fraction increased significantly without changing the K(D) values. SDS-polyacrylamide gel electrophoretic profiles of the 1012-S-eluted fraction showed the existence of two major bands having the molecular weights of approx. 53,000 and 57,000, both of which were irreversibly photoaffinity-labeled with [(3)H]flunitrazepam. This result made a sharp contrast with the result obtained in the rat brain, in which the subunits of the purified receptor consisted of molecular weights of 48,500 and 54,500 and the former band was selectively photoaffinity-labeled with [(3)H]flunitrazepam. In contrast, the gel filtration using Sephadex G-200 of the purified GABA/benzodiazepine receptor complex from both animal species revealed the molecular weight of these receptor complexes was approx. 340,000. These results strongly suggest that the subunit structures of GABA/benzodiazepine receptors from the bovine and rat brains may be essentially different. On the other hand, the purification of GABA/benzodiazepine receptor complex and C1 channel solubilized by CHAPS, a non denaturating zwitterionic detergent, using the same benzodiazepine affinity column followed by DEAE-Sephacel ion exchange column chromatography indicated that the purified GABA receptor was functionally coupled with both benzodiazepine receptor and C1 channel. These results also indicate that GABA(A) receptor which is structurally coupled with benzodiazepine receptor and C1 channel is readily purified by the use of this benzodiazepine affinity gel, whereas GABA(B) receptor which is not associated with both components is recovered in its run-through fraction, respectively.