Characterization of a peripheral‐type benzodiazepine‐binding site in the mitochondria of Chinese hamster ovary cells

Abstract

The isoquinoline carboxamide derivative [³H]PK11195, a ligand for the peripheral-type benzodiazepine (BZD) receptor, binds to Chinese hamster ovary (CHO) cell mitochondria in a specific and saturable manner. Scatchard analysis showed the presence of a single-binding site with an apparent dissociation constant (K _d) of 12.0 ± 1.0 nM and a maximal binding capacity of 23.0 ± 2.0 pmol/mg protein. The pharmacological characterization of this CHO BZD-binding site, based on the displacement of [³H]PK11195 by several drugs of known binding specificity, indicated that it is of the peripheral-type. The photoaffinity probe [³H]PK14105, a nitrophenyl derivative of [³H]PK11195, specifically labeled a 17 kDa CHO mitochondrial protein. This 17 kDa protein was purified from digitonin-solubilized mitochondria by gel-filtration chromatography and two reverse-phase HPLC steps. The purified material migrated as a single band on silver stained or autoradiographed SDS-polyacrylamide gels, and had an amino acid composition corresponding to a 17 kDa protein rich in Leu, Val, Ala, Gly, and Pro. Analysis of the amino-terminal sequence of the purified 17 kDa protein revealed a blocked amino-terminus.