Solubilization and characterization of the liver peripheral-type benzodiazepine receptor

The rat liver membrane-bound and digitonin-solubilized peripheral-type benzodiazepine receptors (mPBZR and dsPBZR, respectively) were characterized. Forty percent of the receptors were solubilized from a liver homogenate with 0.25% digitonin. Scatchard analysis of saturation data for the mPBZR and the dsPBZR showed K(d) = 1.5 nM and maximum number of binding sites = 3.12 pmol/mg of protein and K(d) = 9.2 nM and maximum number of binding sites = 1.10 pmol/mg of protein, respectively. Estimates of K(d) calculated from kinetic data agree with estimates from Scatchard analysis. The affinity of the PBZR for [³H]Ro5-4864 was not affected by guanosine 5'0-(3-thiotriphosphate) which suggests the receptor is not coupled to a G-protein. Competition for specific [³H]Ro5-4864 binding by various ligands demonstrated the same rank order potency of binding inhibition for the membrane bound and solubilized receptors (PK-11195 ≥ Ro5-4864 > diazepam > clonazepam). Thus, the soluble receptor had ligand binding characteristics similar to those of the membrane PBZR. [³H]PK-14105 was used to photoaffinity label the PBZR in a rat liver homogenate. Labeling was specific for the PBZR and the molecular weight of the digitonin-solubilized photoaffinity-labeled receptor was estimated to be 170 kDa by gel filtration chromatography. Estimation of the moelcular weight of the [³H]PK-14105 labeled receptor by sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated a single protein corresponding to 19 kDa.