Some properties of solubilized GABA receptor.
gamma-Aminobutyric acid (GABA) receptor was solubilized from synaptic membrane of the rat brain by various detergents. Nonidet P-40, a non-ionic detergent, was found to be an effective solubilizing agent, since it caused no interference on the receptor binding assay, yielded a [3H]muscimol binding protein with a high specific activity and no aggregation, and preserved good stability of the solubilized fraction. Ammonium sulfate precipitation of the solubilized supernatant significantly increased the binding of [3H]muscimol to GABA receptor, possibly by removing heat-stable and small molecular inhibitory substances. The specific [3H]muscimol binding to the soluble fraction obtained by Nonidet P-40 treatment and subsequent ammonium sulfate precipitation, was saturable with KD 13 and 64 nM, and Bmax 3.4 and 1.8 pmol/mg protein, respectively. The enhancement of the [3H]muscimol binding by diazepam as found in synaptic membrane was also detected in the soluble fraction. Molecular weight of the [3H]muscimol binding site was determined by gel filtration on Sephadex G-200 and was calculated to be 270,000 daltons. This value was identical with that of the [3H]flunitrazepam binding site which appeared in the same solubilized fraction. These results indicate that the properties of solubilized GABA receptor are identical to those of membrane-bound GABA receptor. Furthermore, the present results suggest that both GABA and benzodiazepine receptors may reside on the same macromolecule in synaptic membrane.
D000818:Animals; D001923:Brain Chemistry; D002850:Chromatography, Gel; D003975:Diazepam / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008722:Methods; D008970:Molecular Weight; D009118:Muscimol / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011955:Receptors, Drug / Q000302:isolation & purification; D011963:Receptors, GABA-A; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification* / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D011955:Receptors, Drug; D011963:Receptors, GABA-A; D017981:Receptors, Neurotransmitter; D009118:Muscimol; D005680:gamma-Aminobutyric Acid; D003975:Diazepam