Solubilization of peripheral-type benzodiazepine binding sites from cat cerebral cortex.
The present study demonstrates for the first time the solubilization of peripheral-type benzodiazepine binding sites (PBS) from cat cerebral cortex. Of all detergents tested [digitonin, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholate, and Triton X-100] in the presence of NaCl, the best solubilization (15% of initial activity) was obtained using 0.5% of the zwitterionic detergent CHAPS plus 2 M NaCl. Specific binding of [3H]PK 11195 to membrane-bound and solubilized PBS was saturable, yielding equilibrium dissociation constants (KD) of 1.3 +/- 0.2 and 1.9 +/- 0.3 nM, respectively, and maximal numbers of binding sites of 1,435 +/- 150 and 980 +/- 126 fmol/mg protein, respectively. The KD value of PK 11195 binding to solubilized PBS obtained from experimental kinetic analysis was 0.95 +/- 0.09 nM. The relative potencies of various compounds (PK 11195, Ro 5-4864, diazepam, flunitrazepam, clonazepam, methyl-beta-carboline-3-carboxylate, and Ro 15-1788) in displacing [3H]PK 11195 specific binding from membrane-bound and solubilized PBS were similar. Most of the solubilized binding activity was destroyed by heating at 60 degrees C for 30 min or by treatment with 2 M guanidinium chloride, which indicates the presence of a protein-binding site in the solubilized preparation. Over 85% of the solubilized binding activity was retained after 1 week at 4 degrees C, which will enable future application of purification procedures without major concern for stability of the material.
Awad
M
M
Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa.
Gavish
M
M
D000818:Animals; D001569:Benzodiazepines / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002415:Cats; D002540:Cerebral Cortex / Q000378:metabolism*; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D005260:Female; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D008566:Membranes / Q000378:metabolism; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D007546:Isoquinolines; D001569:Benzodiazepines; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C037850:PK 11195