Solubilization of Peripheral‐Type Benzodiazepine Binding Sites from Cat Cerebral Cortex

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Abstract
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, fluintrazepam, 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.degree. C for 30 min or by treatment with 2 M guanidinium chloride, which indicates the presence of a protein-binding site the solubilized preparation. Over 85% of the solubilized binding activity was retained after 1 week at 4.degree. C, which will enable future application of purification procedures without major concern for stability of the material.

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