title: Solubilization of peripheral benzodiazepine-binding sites from rat kidney.
abstract: 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). KD 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 Bmax values were 4.6 +/- 0.5 and 1.9 +/- 0.2 pmol/mg of protein. The KD 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-beta-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 degrees 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 degrees 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.
mesh_terms: D000818:Animals; D001570:Benzodiazepinones / Q000378:metabolism; D001667:Binding, Competitive; D002243:Carbolines / Q000378:metabolism; D002707:Chlordiazepoxide / Q000378:metabolism; D002998:Clonazepam / Q000378:metabolism; D003975:Diazepam / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D005442:Flumazenil; D005479:Flurazepam / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006358:Hot Temperature; D007546:Isoquinolines / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011963:Receptors, GABA-A / Q000302:isolation & purification*; D012995:Solubility; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
chemical_list: D001570:Benzodiazepinones; D002243:Carbolines; D006146:Guanidines; D007546:Isoquinolines; D011963:Receptors, GABA-A; C026072:1-methyl-beta-carboline-3-carboxylic acid; C028513:4'-chlorodiazepam; D005442:Flumazenil; D002998:Clonazepam; D002707:Chlordiazepoxide; D014508:Urea; D005479:Flurazepam; D019791:Guanidine; D004072:Digitonin; D003975:Diazepam; C037850:PK 11195