A gamma-aminobutyric acid/benzodiazepine receptor complex from bovine cerebral cortex. Improved purification with preservation of regulatory sites and their interactions

• Published in: The Journal of biological chemistry Vol. 259; no. 11; pp. 7219 - 7223
• Main Authors: Sigel, E, Barnard, E A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 10.06.1984; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Biological and medical sciences; Bridged Bicyclo Compounds - metabolism; Bridged Bicyclo Compounds, Heterocyclic; Cattle; Cerebral Cortex - analysis; Cholic Acids; Flunitrazepam - metabolism; gamma-Aminobutyric Acid - pharmacology; Kinetics; Medical sciences; Methods; Muscimol - metabolism; Neuropharmacology; Pentobarbital - pharmacology; Pharmacology. Drug treatments; Psycholeptics: tranquillizer, neuroleptic; Psychology. Psychoanalysis. Psychiatry; Psychopharmacology; Receptors, Cell Surface - isolation & purification; Receptors, GABA-A; Solubility; Amphoteric; Gabaergic receptor; Cerebral cortex; Detergent; Purification; Bovine; Animal; Central nervous system; Brain (Vertebrata); Benzodiazepine receptor; Binding site; Method
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)39860-5

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.