Cloning and expression of cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase.
A cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase (P450cc24) was isolated from a rat kidney cDNA library using specific antibodies to the enzyme. The isolated cDNA was 3.2 kbp long and contained a 1542-bp open reading frame encoding 514 amino acids. The deduced amino acid sequence contained a presequence typical of mitochondrial enzymes in the N-terminal region. The amino acid sequence shows less than 30% similarity to those of any other cytochrome P450s so far reported and, therefore, P450cc24 constitutes a novel family of P450. COS-7 cells transfected with the cDNA produced a protein that was reactive with the antibodies and catalyzed NADPH-dependent 24-hydroxylation of 25-hydroxyvitamin D3 in the presence of adrenodoxin and NADPH-adrenodoxin reductase. Using the cDNA as a probe we demonstrated that the increase of 24-hydroxylation activity caused by administration of vitamin D3 into rats was accompanied by an increase of the mRNA.
Ohyama
Y
Y
Department of Biochemistry, Hiroshima University School of Dentistry, Japan.
Noshiro
M
M
Okuda
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System; D007668:Kidney / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping; D013250:Steroid Hydroxylases / Q000235:genetics*; D014162:Transfection; D065668:Vitamin D3 24-Hydroxylase
D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase