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.

TitleA 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.
Publication TypeJournal Article
Year of Publication1990
AuthorsSu P, Rennert H, Shayiq RM, Yamamoto R, Zheng YM, Addya S, Strauss JF, Avadhani NG
JournalDNA Cell Biol
Volume9
Issue9
Pagination657-67
Date Published1990 Nov
ISSN1044-5498
KeywordsAmino Acid Sequence, Animals, Base Sequence, Blotting, Western, Cholecalciferol, Cholestanetriol 26-Monooxygenase, Cholesterol, Cytochrome P-450 Enzyme System, Female, Gene Expression Regulation, Hydroxylation, Mitochondria, Liver, Molecular Probe Techniques, Molecular Sequence Data, Multienzyme Complexes, Ovary, Rabbits, Rats, Rats, Inbred Strains, RNA, Messenger, Sequence Homology, Nucleic Acid, Steroid Hydroxylases
Abstract

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.

DOI10.1089/dna.1990.9.657
Alternate JournalDNA Cell Biol
PubMed ID2175615
Grant ListCA-22762 / CA / NCI NIH HHS / United States
GM-34883 / GM / NIGMS NIH HHS / United States
HD-06274 / HD / NICHD NIH HHS / United States
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