Species differences and heterogeneity of solubilized peripheral-type benzodiazepine binding sites

The pharmacological characteristics of digitonin-solubilized peripheral-type benzodiazepine binding sites (PBS) from kidney membranes of various species were investigated to determine whether the species differences and heterogeneity observed in membrane-bound binding sites would be maintained after solubilization. [3H]PK 11195 (0.05 to 10 nM) bound with high affinity to rat, guinea pig, calf, and cat kidney solubilized preparations yielding maximal numbers of binding sites (Bmax) of 3,593 +/- 381, 25,645 +/- 1,795, 1,327 +/- 141, and 2,446 +/- 148 fmol/mg protein, respectively, and equilibrium dissociation constant (KD) values of 1.74 +/- 0.18, 2.15 +/- 0.15, 0.85 +/- 0.09, and 1.02 +/- 0.06 nM, respectively. On the other hand, the respective Bmax and KD values for [3H]Ro 5-4864 (1.25 to 40 nM) were 2,688 +/- 275, 14,182 +/- 1,134, 144 +/- 23 and 205 +/- 17 fmol/mg protein (about 75, 55, 11, and 8%, respectively, of that of [3H]PK 11195) and 13.8 +/- 1.5, 14.6 +/- 1.1, 10.6 +/- 1.7, and 19.9 +/- 1.2 nM. Unlabeled Ro 5-4864 was two orders of magnitude more potent in displacing [3H]PK 11195 binding from rat kidney solubilized preparations than from calf kidney solubilized preparations, whereas the potency of unlabeled PK 11195 in displacing [3H]PK 11195 binding from both rat and calf kidney solubilized preparations was almost identical. Analysis of these displacement data revealed that PK 11195 bound to a single population of binding sites (nH approximately equal to 1.0), whereas Ro 5-4864 bound to two populations of binding sites (nH less than 1.0) in both rat and calf kidney solubilized preparations. These results indicate that PBS species differences and heterogeneity observed in membrane-bound binding sites are retained in the soluble state and are probably attributable to variations in the molecular structure of PBS rather than to differences in membrane environment.