Peripheral Benzodiazepine-binding Sites

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 5-4884 to membranebound and solubilized peripheral benzodiazepine-binding sites was saturable, yielding a linear Scatchard plot (r = 0.98). KD values obtained for the membrane-bound and solubilized peripheral benzodiazepine binding sites were 3.9 f 0.4 nM and 5.4 + 0.4 nM, respectively. Respective B,.. values were 4.6 f 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, Fio 54864, diazepam, flurazepam, chlordiazepoxide, Ro 15-1788, methyl-8-carboline-&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 4X, and more than 60% was retained after 4 days at the same temperature. These results indicate that the binding characteristics of peripheral benzodiazepinebinding sites extant in the membrane-bound state are retained after solubilization. The discovery of high affinity benzodiazepine (BZ)-binding sites in the CNS (Squires and Braestrup, 1977; Mohler and Okada, 1977) promoted studies which have shed light on the understanding of the GABA/BZ receptor complex. It has been found that the binding of various BZs to these sites correlates with their clinical potency as anticonvulsants and anxiolytics (Mohler et al., 1978). The solubilization of the central BZ receptors (Gavish et al., 1979; Lang et al., 1979; Yousufi et al., 1979; Sherman-Gold and Dudai, 1980; Stephenson and Olsen, 1982) was an important step in the characterization (Gavish and Snyder, 1980; Chang et al., 1982; Gavish, 1983) Received October 22, 1984; Revised February 21, 1985; Accepted March 19, 1985 ’ This work was supported by grants from the M. Hedson Foundation for MedIcal Research and the Bat-Sheva de Rothschild Foundation. ‘To whom correspondence should be addressed, at Rappaporl Family Institute for Research in the Medical Sciences and Department of Pharmacology, Faculty of Medicine, Technlon-Israel Institute of Technology, P. 0. Box 9697, Haifa 31096, Israel. and partial purification (Gavish and Snyder, 1981; Martini et al., 1982; Schoch and Mohler, 1983; Sigel and Barnard, 1984) of the GABA/ BZ receptor complex. In addition to the “central” BZ receptors mentioned above, another type of BZ-binding site has been located, initially in peripheral tissues such as platelets (Wang et al., 1980), mast cells (Taniguchi et al., 1980), thymocytes (Wang et al., 1981), heart (Davies and Huston, 1981; Taniguchi et al., 1982), and kidney (Taniguchi et al., 1982), but also in the brain (Schoemaker et al., 1981; Marangos et al., 1982; Weissman et al., 1984). Peripheral binding sites are different from central BZ receptors in their distribution within the brain, their specificity for ligand binding, and their lack of coupling to the GABA receptor. As a first step toward purification of peripheral BZ-binding sites, we conducted solubilization experiments on rat kidney, which is rich in peripheral BZ-binding sites which are similar in their drug specificity to the peripheral BZbinding sites in the brain. Active Triton X-100.solubilized peripheral BZ-binding sites, after detergent removal, have already been demonstrated (Martini et al., 1983). The experiments undertaken here demonstrate detailed characterization of digitonin-solubilized peripheral BZ-binding sites from rat kidney without prior removal of the detergent. Materials and Methods Materials. [3H]Ro 5-4864 was purchased from New England Nuclear (Boston, MA). Unlabeled BZs were kindlv suoolied bv Drs. H. Gutmann and i. Kyburz, Hbffman-La Roche (Basel, Sditze%nd). Unlabeled PK 11195 was a generous gift from Dr. G. Le Fur (Pharmuka Laboratoires, Gennevilliers, France). 3-[(3-Chloramidopropyl)dlmethylammonio]-1 -propanesulfonate (CHAPS) was obtained from Calbiochem (La Jolla, CA). Digltonin, deoxycholate (DOC), and Triton X-100 were purchased from Sigma Chemical Co. (St. Louis, MO). All other compounds were purchased from commercial sources. Membrane solubikation. Male Sprague-Dawley rats were decapitated, and their kidneys were removed and frozen at -20°C. The kidney (1 qm) was defrosted and homogenized in 50 vol of Tns-HCI buffer (pi 7.4) ai 4°C with a Brinkman Polvtron (settina IO) for 15 sec. The homoaenate was centrifuged at 49,000 X b for 15 miqand’the pellet was either homogenized In 60 vol of 50 mM Tris-HCI buffer (pH 7.4) and used for binding studies or solubilized by detergents. The pellet of washed membranes was homogenized in 60 vol of 50 mM Tns-HCI buffer (pH 7.4) containing detergents at different concentrations and was stirred for 30 mln at 4°C. A portion of this material was assayed for binding activity, and the rest was centrifuged for 60 min at 100,000 x g. The resultant supernatant was diluted 1:l and used as a soluble preparatjon, and the pellet was rehomogenlzed In the original volume of Tris-HCI buffer and tested for binding activity. Binding assay. Binding acttvity of peripheral BZ-binding sites was assayed In 50 mM Tns-HCI buffer (pH 7.4) in a final volume of 500 UI containina 400 /LI of soluble or membrane peripheral BZ-binding sites (80 to 160 ig of protein) and 50 ~1 of [3H]Ro 5-4864 (0.5 to 40 nM final concentration), in the absence (total binding) or presence (nonspecific binding) of 1 FM unlabeled Ro 5-4864 or 1 PM unlabeled PK 11195. After incubation for 60 min at 4”C, 2890 Gavish and Fares Vol. 5, No. 11, Nov. 1985 samples were filtered under vacuum over Whatman GF/B filters, treated with polyethyleneimrne (Bruns et al., 1983) and washed three times with 5 ml of 50 mM Tris-HCI buffer (pH 7.4). Filters were placed in vials and counted for radioactivity.