Araya Z, Hellman U, Hansson R
Department of Pharmaceutical Biosciences, University of Uppsala, Sweden.
Eur J Biochem. 1995 Aug 1;231(3):855-61. doi: 10.1111/j.1432-1033.1995.0855d.x.
A fraction of cytochrome P-450 catalysing an efficient 6 alpha-hydroxylation of taurine-conjugated 3 alpha,7 alpha-dihydroxy-5 beta- cholanoic acid (taurochenodeoxycholic acid) was partially purified from pig liver microsomes. The specific content of cytochrome P-450 was 6 nmol/mg protein and the preparation showed two major protein bands upon SDS/PAGE. These two bands were isolated after SDS/PAGE and protein blotting. The protein band with a molecular mass of 53 kDa had an N-terminal amino acid sequence and internal sequences resembling that of the cytochrome P-450 4A subfamily (CYP 4A). Polyclonal antibodies raised against this protein were able to, after SDS/PAGE and immunoblotting, detect the protein in microsomal fractions as well as in the purified cytochrome P-450 fraction. Furthermore, addition of these antibodies to a reconstituted system containing the cytochrome P-450 fraction, inhibited 6 alpha-hydroxylation of taurochenodeoxycholic acid by up to 90%. Experiments with irrelevant antibodies did not show inhibition of 6 alpha-hydroxylation. The purified cytochrome P-450 fraction catalysed in addition omega- and omega-1 hydroxylation of lauric acid and 6 alpha-hydroxylation of 3 alpha-hydroxy-5 beta-cholanoic acid (lithocholic acid). However, these hydroxylase activities were rather low compared to 6 beta-hydroxylation of taurochenodexycholic acid. The enzyme fraction did not show hydroxylase activities towards cholesterol and 5 beta-cholestane-3 alpha,7 alpha-diol. These results indicate that 6 alpha-hydroxylation of taurochenodeoxycholic acid is catalysed by a specific species of cytochrome P-450 that, according to N-terminal amino acid sequence as well as catalytic properties, could be a member of the CYP 4A subfamily.
从猪肝微粒体中部分纯化出了一小部分能高效催化牛磺酸结合的3α,7α-二羟基-5β-胆烷酸（牛磺鹅去氧胆酸）6α-羟基化反应的细胞色素P-450。细胞色素P-450的比含量为6 nmol/mg蛋白质，该制剂在SDS/PAGE上显示出两条主要蛋白带。这两条带在SDS/PAGE和蛋白质印迹后被分离出来。分子量为53 kDa的蛋白带具有与细胞色素P-450 4A亚家族（CYP 4A）相似的N端氨基酸序列和内部序列。针对该蛋白产生的多克隆抗体在SDS/PAGE和免疫印迹后，能够在微粒体组分以及纯化的细胞色素P-450组分中检测到该蛋白。此外，将这些抗体添加到含有细胞色素P-450组分的重组系统中，可使牛磺鹅去氧胆酸的6α-羟基化反应受到高达90%的抑制。用无关抗体进行的实验未显示对6α-羟基化反应的抑制作用。纯化的细胞色素P-450组分还催化了月桂酸的ω-和ω-1羟基化反应以及3α-羟基-5β-胆烷酸（石胆酸）的6α-羟基化反应。然而，与牛磺鹅去氧胆酸的6β-羟基化反应相比，这些羟化酶活性相当低。该酶组分对胆固醇和5β-胆甾烷-3α,7α-二醇未显示出羟化酶活性。这些结果表明，牛磺鹅去氧胆酸的6α-羟基化反应由一种特定的细胞色素P-450催化，根据N端氨基酸序列以及催化特性，它可能是CYP 4A亚家族的一员。
