title: Solubilization and characterization of the liver peripheral-type benzodiazepine receptor.
abstract: 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 Kd = 1.5 nM and maximum number of binding sites = 3.12 pmol/mg of protein and Kd = 9.2 nM and maximum number of binding sites = 1.10 pmol/mg of protein, respectively. Estimates of Kd 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 greater than or equal to Ro5-4864 greater than diazepam greater than 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 molecular weight of the [3H]PK-14105 labeled receptor by sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated a single protein corresponding to 19 kDa.
authors.affiliation: Department of Pharmacology & Toxicology, College of Pharmacy, University of Arizona, Tucson.
authors.forename: A L
authors.initials: AL
authors.lastname: Parola
authors.forename: C W
authors.initials: CW
authors.lastname: Putnam
authors.forename: D H
authors.initials: DH
authors.lastname: Russell
authors.forename: H E
authors.initials: HE
authors.lastname: Laird
mesh_terms: D000818:Animals; D001570:Benzodiazepinones / Q000378:metabolism; D001667:Binding, Competitive; D002850:Chromatography, Gel; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D066298:In Vitro Techniques; D008099:Liver / Q000032:analysis* / Q000502:physiology; D008970:Molecular Weight; D051381:Rats; D011963:Receptors, GABA-A / Q000302:isolation & purification* / Q000502:physiology; D012995:Solubility; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D001570:Benzodiazepinones; D011963:Receptors, GABA-A; D013873:Thionucleotides; C028513:4'-chlorodiazepam; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate