title: Identification of a novel rat microsomal vitamin D3 25-hydroxylase.
abstract: Vitamin D3 requires the 25-hydroxylation in the liver and the subsequent 1alpha-hydroxylation in the kidney to exert its biological activity. Vitamin D3 25-hydroxylation is hence an essential modification step for vitamin D3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D3 relative to other substrates. In this study, we purified vitamin D3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D3 and 1alpha-hydroxyvitamin D3 were 1.1 and 13 nmol/min/nmol of P450, respectively. The purified fraction showed a few protein bands in a 50-60-kDa range on SDS-PAGE, typical for a cytochrome P450. The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D3 and 1alpha-hydroxyvitamin D3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases. Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D3 25-hydroxylation in rat liver.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Yamasaki
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Izumi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ide
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Ohyama
mesh_terms: D000818:Animals; D053493:Cholestanetriol 26-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism*; D005260:Female; D006900:Hydroxylation; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009928:Organ Specificity; D010105:Oxygenases / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012737:Sex Factors; D013250:Steroid Hydroxylases / Q000032:analysis / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D013250:Steroid Hydroxylases; C106098:Cyp2j3 protein, rat; D053493:Cholestanetriol 26-Monooxygenase