The ability of a variety of detergents to solubilize peripheral benzodiazepine-binding sites from rat kidney was tested. Of all the detergents tested, only digitonin was found to be suitable for solubilization. This detergent solubilized 21% of the binding activity; 47% was inactivated, and 32% remained in the pellet. Specific binding of [3H]Ro 5-4864 to membrane-bound and solubilized peripheral benzodiazepine-binding sites was saturable, yielding a linear Scatchard plot (r = 0.96). K(D) values obtained for the membrane-bound and solubilized peripheral benzodiazepine binding sites were 3.9 ± 0.4 nM and 5.4 ± 0.4 nM, respectively. Respective B(max) values were 4.6 ± 0.5 and 1.9 ± 0.2 pmol/mg of protein. The K(D) value for the solubilized material obtained from kinetic experiments was 5.3 ± 0.6 nM. The potency of PK 11195, Ro 5-4864, diazepam, flurazepam, chlordiazepoxide, Ro 15-1788, methyl-β-carboline-3-carboxylate, and clonazepam to displace bound [3H]Ro 5-4864 from peripheral binding sites was similar in the membrane-bound and the soluble states. Most of the binding activity of the solubilized binding sites was destroyed by heating at 60°C for 30 min or by treatment with 2 M guanidinium chloride or 4 M urea. More than 95% of the binding activity of the solubilized binding sites was retained after 18 hr at 4°C, and more than 60% was retained after 4 days at the same temperature. These results indicate that the binding characteristics of peripheral benzodiazepine-binding sites extant in the membrane-bound state are retained after solubilization.
|Number of pages||5|
|Journal||Journal of Neuroscience|
|State||Published - 1985|
ASJC Scopus subject areas
- Neuroscience (all)