Cofractionation of the 17-kD PK 14105 binding site protein with solubilized peripheral-type benzodiazepine binding...

To examine the relationship between PKBS, a 17-kD protein covalently photolabeled by [3H]PK 14105, and its association with peripheral-type benzodiazepine binding sites, rat adrenal mitochondrial fractions were photolabeled with [3H]PK 14105, solubilized in digitonin, and subjected to anion-exchange chromatography over Q-Sepharose. The chromatographic behavior of PKBS was evident as principally two major fractions, signified as Q-I and Q-II. Specific binding sites for [3H]Ro5-4864 and [3H]PK 11195 were also assayed and found to cofractionate with each other and in a manner which coincided with the photolabeled PKBS profile. The Q-I and Q-II fractions were further distinguished based on their different molecular sizes observed by gel filtration, yet both fractions were characterized as containing peripheral-type benzodiazepine recognition sites according to the following criteria. Scatchard analysis of both subpopulations revealed a single class of binding sites for [3H]Ro5-4864 with an apparent KD of 14 nM for Q-I and 22 nM for Q-II; these affinities were slightly lower than those found in mitochondrial membrane preparations used as the starting material for solubilization. The rank order of potency to inhibit [3H]Ro5-4864 binding in both subpopulations was PK 11195 greater than Ro5-4864 greater than diazepam greater than clonazepam, in connection with the pharmacological specificity of membrane-associated peripheral-type benzodiazepine binding sites. These studies provide direct biochemical evidence that the recognition sites for benzodiazepines and isoquinoline carboxamides cofractionate in unison with the 17-kD PKBS protein, demonstrating an intimate relationship between this protein and the binding domains for peripheral-type benzodiazepine ligands.