A gamma-aminobutyric acid/benzodiazepine receptor complex from bovine cerebral cortex. Improved purification with preservation of regulatory sites and their interactions.
A gamma-aminobutyrate/benzodiazepine receptor complex has been purified from bovine cerebral cortex by an improved procedure using a zwitterionic detergent. A high affinity binding site for the chloride ion channel-blocking ligand [35S]t-butyl bicyclophosphorothionate ( TBPS ) was co-purified with the high affinity binding sites for gamma-aminobutyrate and benzodiazepines. The latter two have previously been shown to reside on the same physical structure ( Sigel , E., Stephenson , F.A., Mamalaki , C., and Barnard , E. A. (1983) J. Biol. Chem. 258, 6965-6971). The dissociation constants, as measured in assay medium containing zwitterionic detergent were 90 +/- 20 nM for TBPS and 11 +/- 4 nM for [3H]flunitrazepam, whereas the binding of [3H]muscimol, a gamma-aminobutyrate agonist, showed a more complex binding behavior with more than one site. If the same preparation was assayed in a medium containing instead Triton X-100 as the detergent, the binding of TBPS was strongly inhibited, [3H]flunitrazepam binding was unaffected, and [3H]muscimol bound to a single class of sites with a dissociation constant of 33 +/- 3 nM. Regulatory interactions were retained in the complex isolated by the improved method: [3H]flunitrazepam binding was stimulated by gamma-aminobutyrate or by pentobarbital, and in a dose-dependent manner. The same two subunit types of Mr = 53,000 and 57,000 are present in the purified receptor complex as previously reported.
D000818:Animals; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002417:Cattle; D002540:Cerebral Cortex / Q000032:analysis*; D002793:Cholic Acids; D005445:Flunitrazepam / Q000378:metabolism; D007700:Kinetics; D008722:Methods; D009118:Muscimol / Q000378:metabolism; D010424:Pentobarbital / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D011963:Receptors, GABA-A; D012995:Solubility; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002793:Cholic Acids; D011956:Receptors, Cell Surface; D011963:Receptors, GABA-A; D009118:Muscimol; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam; C037476:tert-butylbicyclophosphorothionate; D010424:Pentobarbital; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate