Identification of a novel rat microsomal vitamin D3 25-hydroxylase

被引:44
作者
Yamasaki, T [1 ]
Izumi, S [1 ]
Ide, H [1 ]
Ohyama, Y [1 ]
机构
[1] Hiroshima Univ, Dept Math & Life Sci, Grad Sch Psychol, Higashihiroshima 7398526, Japan
关键词
D O I
10.1074/jbc.M311346200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Vitamin D-3 requires the 25-hydroxylation in the liver and the subsequent 1alpha-hydroxylation in the kidney to exert its biological activity. Vitamin D-3 25-hydroxylation is hence an essential modification step for vitamin D-3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D-3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D-3 relative to other substrates. In this study, we purified vitamin D-3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D-3 and 1alpha-hydroxyvitamin D-3 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 D-3 and 1alpha-hydroxyvitamin D-3 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 D-3 25-hydroxylation in rat liver.
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页码:22848 / 22856
页数:9
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