Yamasaki Tomoaki, Izumi Shunsuke, Ide Hiroshi, Ohyama Yoshihiko
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
J Biol Chem. 2004 May 28;279(22):22848-56. doi: 10.1074/jbc.M311346200. Epub 2004 Mar 16.
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.
维生素D3需要先在肝脏中进行25-羟化，随后在肾脏中进行1α-羟化才能发挥其生物活性。因此，维生素D3的25-羟化是维生素D3激活的一个关键修饰步骤。到目前为止，已有三种细胞色素P450分子类型（CYP27A1、CYP2C11和CYP2D25）被明确鉴定为维生素D3 25-羟化酶。然而，由于它们具有广泛的底物特异性且相对于其他底物而言对维生素D3的活性较低，其生理作用仍不清楚。在本研究中，我们从雌性大鼠肝脏微粒体中纯化了维生素D3 25-羟化酶。纯化组分对维生素D3和1α-羟基维生素D3的活性分别为1.1和13 nmol/分钟/每nmol P450。在SDS-PAGE上，纯化组分在50 - 60 kDa范围内显示出几条蛋白带，这是细胞色素P450的典型特征。利用基质辅助激光解吸电离/飞行时间质谱对一条蛋白带（56 kDa）进行胰蛋白酶肽质量指纹图谱分析，确定该条带为CYP2J3。CYP2J3在大肠杆菌中进行了异源表达。纯化的重组CYP2J3对维生素D3和1α-羟基维生素D3表现出很强的25-羟化活性，转换数分别为3.3和22，明显高于先前鉴定为25-羟化酶的P450。定量PCR分析表明，CYP2J3 mRNA的表达水平与CYP27A1相似，且无明显的性别差异。这些结果有力地表明，CYP2J3是大鼠肝脏中负责维生素D3 25-羟化的主要P450。
