Axén E, Bergman T, Wikvall K
Department of Pharmaceutical Biochemistry, University of Uppsala, Sweden.
Biochem J. 1992 Nov 1;287 ( Pt 3)(Pt 3):725-31. doi: 10.1042/bj2870725.
A cytochrome P-450 which catalyses 25-hydroxylation of vitamin D3 has been purified to apparent homogeneity from pig liver microsomes. The specific content of cytochrome P-450 was 12 nmol.mg of protein-1, and the preparation showed a single band with an apparent M(r) of 50,500 upon SDS/PAGE. A monoclonal antibody raised against the vitamin D3 25-hydroxylase reacted strongly with the purified 25-hydroxylating cytochrome P-450 from pig kidney microsomes [Bergman & Postlind (1990) Biochem. J. 270, 345-350]. The liver enzyme showed structural and functional properties very similar to those of the kidney enzyme. The two enzymes differed with respect to only one of the first 16 N-terminal amino acids. The vitamin D3 25-hydroxylase in pig liver microsomes exhibited a turnover and an apparent Km for 25-hydroxylation of vitamin D3 which were of the same order of magnitude as those of a well-characterized male-specific 25-hydroxylating cytochrome P-450 in rat liver microsomes. The two enzymes differed structurally. The pig liver enzyme was, in contrast to the rat liver enzyme, not sex-specific, and did not catalyse 16 alpha-hydroxylation of testosterone. These properties of the 25-hydroxylase in rat liver microsomes have led to questions on the role of microsomal 25-hydroxylation of vitamin D3. It is concluded that studies on microsomal 25-hydroxylation with the rat may be misleading. The results of the present study show that the pig appears to be a representative species for evaluation of vitamin D3 hydroxylases in other mammals, including man.
一种催化维生素D3 25-羟化反应的细胞色素P-450已从猪肝微粒体中纯化至表观均一。细胞色素P-450的比含量为12 nmol·mg蛋白质-1，该制剂在SDS/PAGE上呈现出一条表观相对分子质量为50500的单带。针对维生素D3 25-羟化酶产生的单克隆抗体与从猪肾微粒体中纯化的25-羟化细胞色素P-450发生强烈反应[伯格曼和波斯特林德（1990年）《生物化学杂志》270卷，345 - 350页]。肝脏酶显示出与肾脏酶非常相似的结构和功能特性。这两种酶在前16个N端氨基酸中只有一个不同。猪肝微粒体中的维生素D3 25-羟化酶对维生素D3 25-羟化反应的转换率和表观Km与大鼠肝微粒体中一种特性明确的雄性特异性25-羟化细胞色素P-450的转换率和表观Km处于同一数量级。这两种酶在结构上有所不同。与大鼠肝脏酶不同，猪肝酶不是性别特异性的，也不催化睾酮的16α-羟化反应。大鼠肝微粒体中25-羟化酶的这些特性引发了关于维生素D3微粒体25-羟化作用的疑问。得出的结论是，用大鼠进行的微粒体25-羟化研究可能会产生误导。本研究结果表明，猪似乎是评估包括人类在内的其他哺乳动物中维生素D3羟化酶的代表性物种。
