Characterization of a peripheral-type benzodiazepine-binding site in the mitochondria of Chinese hamster ovary cells.
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 (Kd) 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.
Riond
J
J
Sanofi Elf Bio-Recherches, Labège, France.
Vita
N
N
Le Fur
G
G
Ferrara
P
P
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007546:Isoquinolines / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010053:Ovary; D010777:Photochemistry; D011963:Receptors, GABA-A / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D000596:Amino Acids; D007546:Isoquinolines; D011963:Receptors, GABA-A; C051238:PK 14105; C037850:PK 11195