Purification of gamma-aminobutyric acid (GABA) receptor from rat brain by affinity column chromatography using a new benzodiazepine, 1012-S, as an immobilized ligand.
The purification of gamma-aminobutyric acid (GABA) and benzodiazepine receptors from the rat brain was employed by the affinity column using a new benzodiazepine, 1012-S, as immobilized ligand. The 1012-S has a aliphatic primary amino group and exhibited an extremely high potency for displacing [3H]flunitrazepam binding to solubilized benzodiazepine receptor preparation (IC50 = 6.0 X 10(-11) M). This benzodiazepine affinity gel retained almost all of the solubilized GABA receptors from synaptic membranes applied to the column, and 25.6% of the receptor was eluted bio-specifically following the application of 1 mM 1012-S. The highest purification fold thus obtained was 4576 (specific activity: 0.99 nmol/mg protein). Furthermore, the successive application of 1-2 M NaSCN also resulted the elution of a highly enriched GABA receptor (specific activity: 0.41 nmol/mg protein; purification fold: 1889). SDS-polyacrylamide gel electrophoretic profiles of the bio-specifically eluted fraction with 1012-S showed the existence of two major bands having the molecular weights of approximately 48,500 and 54,500, in which the former band was selectively photoaffinity-labeled with [3H]flunitrazepam. On the other hand, it was found that the non-specifically eluted fraction with NaSCN contained 4 additional minor bands having molecular weights of 41,000 to 51,000. These results indicate that GABA receptor of the rat brain is coupled, at least in part, with benzodiazepine receptor and is readily purified by the use of highly specific benzodiazepine affinity gel, 1012-S-acetamide adipic hydrazide Sepharose 4B. The present results also suggest that the purified GABA/benzodiazepine receptor complex may contain two different kinds of subunits having the molecular weights of 48,000 and 54,500, in which the former subunit may possess benzodiazepine binding sites.
D000818:Animals; D001569:Benzodiazepines; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008024:Ligands; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D011963:Receptors, GABA-A / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives
D008024:Ligands; D011963:Receptors, GABA-A; C044711:1012-S-acetamide adipic hydrazide Sepharose 4B; D001569:Benzodiazepines; D012685:Sepharose