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J. Biol. Chem., Vol. 279, Issue 22, 22848-22856, May 28, 2004

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Identification of a Novel Rat Microsomal Vitamin D₃ 25-Hydroxylase^*

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

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

Vitamin D₃ requires the 25-hydroxylation in the liver and the subsequent 1-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 1-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 1-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.

Received for publication, October 15, 2003 , and in revised form, January 26, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains two additional figures and two additional tables.

To whom correspondence should be addressed: Dept. of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan. Tel./Fax: 81-82-424-7458; E-mail: ohyama{at}sci.hiroshima-u.ac.jp.

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