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Characterization of a peripheral‐type benzodiazepine‐binding site in the mitochondria of Chinese hamster ovary cells
Author(s) -
Riond J.,
Vita N.,
Le Fur G.,
Ferrara P.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)80229-7
Subject(s) - chinese hamster ovary cell , digitonin , dissociation constant , photoaffinity labeling , biochemistry , peptide sequence , chemistry , microbiology and biotechnology , peptide , hamster , receptor , amino acid , mitochondrion , biology , enzyme , gene
The isoquinoline carboxamide derivative [ 3 H]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 [ 3 H]PK11195 by several drugs of known binding specificity, indicated that it is of the peripheral‐type. The photoaffinity probe [ 3 H]PK14105, a nitrophenyl derivative of [ 3 H]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.