Binding sites for muscimol and flunitrazepam were solubilized and their properties studied by ultrafiltration, isoelectro-focusing and cation-exchange chromatography methods. The heterogenity of binding sites for flunitrazepam was demonstrated in the ultrafiltration experiments: the binding sites with a molecular weight of more than 300,000 dalton were sensitive to stimulation by gamma-aminobutyric acid (GABA) and those with a molecular weight of less than 300,000 dalton were not. The binding sites for flunitrazepam appeared to be low acidic molecules with pI 5.6-6.0 and binding sites for muscimol an apparently more heterogenous distribution in the range of pH 4.6 to 5.9 was detected. Cation-exchange chromatography revealed two subpopulations of binding sites for muscimol one of which copurified together with binding sites for flunitrazepam. Each subpopulation exhibited only one class of binding site with Kd = 20 nM for a subpopulation copurifying together with binding sites for flunitrazepam and Kd = 9 nM for the other.
Animals Brain