Purification and identification of an estrogen binding protein from rat brain: oligomycin sensitivity-conferring protein (OSCP), a subunit of mitochondrial F0F1-ATP synthase/ATPase

• Published in: The Journal of steroid biochemistry and molecular biology Vol. 68; no. 1; pp. 65 - 75
• Main Authors: Zheng, Jianbiao, Ramirez, Victor D.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 1999; Elsevier Science
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - isolation & purification; Adenosine Triphosphatases - metabolism; Amino Acid Sequence; Animals; Binding, Competitive; Biochemistry and metabolism; Biological and medical sciences; Brain - metabolism; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Cattle; Central nervous system; Estrogens - metabolism; Female; Fundamental and applied biological sciences. Psychology; In Vitro Techniques; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Mitochondria - metabolism; Molecular Sequence Data; Oligomycins - metabolism; Rats; Rats, Sprague-Dawley; Receptors, Estrogen - metabolism; Serum Albumin, Bovine; Uterus - metabolism; Vertebrates: nervous system and sense organs; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; B, whole brain without cerebellum and olfactory bulb; DES, diethylstilbestrol; P2, mitochondrial-synaptosomal membrane fraction; PVDF, polyvinylidene difluoride; K i, inhibition constant; PEI, polyethylenimine; M r, molecular weight; mP2, mitochondrial fraction; PBS, phosphate buffer solution; BSA, bovine serum albumin; 17β-E-6-BSA, 17β-estradiol 6-(O-carboxymethyl)oxime-BSA; rbOSCP, recombinant bovine OSCP; B max, binding sites; 17α-E-6-BSA, 17α-estradiol 6-(O-carboxymethyl)oxime-BSA; IC 50, half-inhibition concentration; 17β-E-6-CMO, 17β-estradiol 6-(O-carboxymethyl)oxime; CB, cerebellum; OSCP, oligomycin sensitivity-conferring protein; 17β-E-17-BSA, 17β-estradiol 17-hemisuccinate-BSA; T-3-BSA, testosterone 3-(O-carboxymethyl)oxime-BSA; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; nER, nuclear estrogen receptors; 17β-E-6-[ 125I]BSA, [ 125I]-labeled 17β-E-6-BSA; DCC, dextran-coated charcoal, K d, dissociation constant; 17β-E-17-HS, 17β-estradiol 17-hemisuccinate; Purification; Rat; Rodentia; Central nervous system; Estrogen; Subunit; Ovarian hormone; Characterization; Binding protein; Vertebrata; Mitochondria; Mammalia; Sex steroid hormone; Brain (vertebrata)
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/S0960-0760(98)00161-7

Early studies have suggested the presence in the central nervous system of possible estrogen binding sites/proteins other than classical nuclear estrogen receptors (nER). We report here the isolation and identification of a 23 kDa membrane protein from digitonin-solubilized rat brain mitochondrial fractions that binds 17β-estradiol conjugated to bovine serum albumin at C-6 position (17β-E-6-BSA), a ligand that also specifically binds nER. This protein was partially purified using affinity columns coupled with 17β-E-6-BSA and was recognized by the iodinated 17β-E-6-BSA (17β-E-6-[ 125I]BSA) in a ligand blotting assay. The binding of 17β-E-6-BSA to this protein was specific for the 17β-estradiol portion of the conjugate, not BSA. Using N-terminal sequencing and immunoblotting, this 23 kDa protein was identified as the oligomycin-sensitivity conferring protein (OSCP). This protein is a subunit of the F0F1 (F-type) mitochondrial ATP synthase/ATPase required for the coupling of a proton gradient across the F0 sector of the enzyme in the mitochondrial membrane to ATP synthesis in the F1 sector of the enzyme. Studies using recombinant bovine OSCP (rbOSCP) in ligand blotting revealed that rbOSCP bound 17β-E-6-[ 125I]BSA with the same specificity as the purified 23 kDa protein. Further, in a ligand binding assay, 17β-E-6-[ 125I]BSA also bound rbOSCP and it was displaced by both 17β-E-6-BSA and 17α-E-6-BSA as well as partially by 17β-estradiol and diethylstilbestrol (DES), but not by BSA. This finding opens up the possibility that estradiol, and probably other compounds with similar structures, in addition to their classical genomic mechanism, may interact with ATP synthase/ATPase by binding to OSCP, and thereby modulating cellular energy metabolism. Current experiments are addressing such an issue.