Characterization of a female-specific hepatic mitochondrial cytochrome P-450 whose steady-state level is modulated by testosterone.
Polyclonal antibody to mitochondrial P-450c27/25 reacted with two proteins of apparent molecular masses of 52 kilodaltons (kDa) and 50 kDa from the female rat liver mitochondrial proteins bound to an omega-octylaminoagarose column. The two proteins were purified to greater than 85% homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography, and both were found to be P-450 as judged by dithionite-reduced CO difference spectra. Both of the P-450 forms required mitochondrial-specific ferredoxin and ferredoxin reductase for in vitro reconstitution of enzyme activities, suggesting that they are mitochondrial forms. The 52-kDa P-450 exhibited the properties of mitochondrial 27/25-hydroxylase with respect to high vitamin D3 25-hydroxylase activity [1.4 nmol (nmol of P-450)-1 min-1] and N-terminal amino acid sequence. The 50-kDa P-450, on the other hand, lacked significant vitamin D3 25-hydroxylase activity, but showed 17 beta-reductase [0.380-0.400 nmol (nmol of P-450)-1 min-1] and 17 beta-oxidase [0.1-0.16 nmol (nmol of P-450)-1 min-1] activities with both androgens and estrogens as substrates. Immunoblot analysis of proteins using a monoclonal antibody specific for P-450c27/25 showed a 2-3-fold higher level of this enzyme in the female liver mitochondria than in the males. Similarly, use of a polyclonal antibody in the immunoblot analysis showed that the 50-kDa P-450 is female-specific. The relative level of P-450c27/25 was reduced significantly in castrated females, while the level of the female-specific 50-kDa P-450 was increased. However, the levels of both enzymes were increased in castrated males.(ABSTRACT TRUNCATED AT 250 WORDS)
Addya
S
S
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104-6048.
Zheng
Y M
YM
Shayiq
R M
RM
Fan
J Y
JY
Avadhani
N G
NG
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D004285:Dogs; D005260:Female; D007700:Kinetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D012727:Sex Characteristics; D013379:Substrate Specificity; D013739:Testosterone / Q000494:pharmacology*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System