A cDNA encoding a rat mitochondrial cytochrome P450 catalyzing both the 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3: gonadotropic regulation of the cognate mRNA in ovaries.
A cDNA expression library prepared from rat liver RNA was screened with a polyclonal antibody specific for mitochondrial vitamin D3 25-hydroxylase and a cDNA for rabbit liver mitochondrial cytochrome P450c26 (CYP 26), yielding cDNA clones with identical sequences. The deduced amino acid sequence derived from a 1.9-kb full-length cDNA was 73% identical to that of rabbit cytochrome P450c26. A monoclonal antibody was used to demonstrate that the product of the 1.9-kb cDNA clone was targeted to the mitochondrial compartment when expressed in COS cells. Mitochondrial membranes containing the expressed protein showed both vitamin D3 25-hydroxylase and cholesterol 26-hydroxylase activities when reconstituted with ferredoxin reductase and ferredoxin, demonstrating that the same P450, designated as P450c26/25, can catalyze both reactions. Northern blot analysis revealed that the P450c26/25 cDNA hybridizes with a 2.4-kb RNA from rat liver and unstimulated ovaries. Treatment of rats with pregnant mare's serum gonadotropin resulted in a fivefold increase in the 2.4-kb mRNA as well as the appearance of a 2.1-kb mRNA species in the ovaries. Our findings document the presence of a regulated bifunctional mitochondrial cytochrome P450 capable of catalyzing the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of cholesterol.
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Department of Animal Biology, University of Pennsylvania, School of Veterinary Medicine, Philadelphia 19104.
Rennert
H
H
Shayiq
R M
RM
Yamamoto
R
R
Zheng
Y M
YM
Addya
S
S
Strauss
J F
JF
Avadhani
N G
NG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002762:Cholecalciferol / Q000378:metabolism; D053493:Cholestanetriol 26-Monooxygenase; D002784:Cholesterol / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006900:Hydroxylation; D008930:Mitochondria, Liver / Q000201:enzymology*; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D010053:Ovary / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D012689:Sequence Homology, Nucleic Acid; D013250:Steroid Hydroxylases / Q000235:genetics* / Q000378:metabolism
D009097:Multienzyme Complexes; D012333:RNA, Messenger; D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase