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M311346200v1
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Papers In Press, published online ahead of print March 16, 2004
J. Biol. Chem, 10.1074/jbc.M311346200
Submitted on October 15, 2003
Revised on March 16, 2004
Accepted on March 16, 2004

Identification of a novel rat microsomal vitamin D3 25-hydroxylase

Tomoaki Yamasaki, Shunsuke Izumi, Hiroshi Ide, and Yoshihiko Ohyama

Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8526

Corresponding Author: ohyama{at}sci.hiroshima-u.ac.jp

Vitamin D3 requires the 25-hydroxylation in the liver and the subsequent 1a-hydroxylation in the kidney to exert its biological activity. Vitamin D3 25-hydroxylation is hence an essential modification step for vitamin D3 activation. Until today, three cytochrome P450 molecular species (CYP27A1, CYP2C11, 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 1a-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 identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D3 and 1a-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 in similar level to that of CYP27A1 without striking sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D3 25-hydroxylation in rat liver.


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