Chang T K, Teixeira J, Gil G, Waxman D J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Biochem J. 1993 Apr 15;291 ( Pt 2)(Pt 2):429-33. doi: 10.1042/bj2910429.
CYP 3A10 is a hamster liver cytochrome P-450 (P450) that encodes lithocholic acid 6 beta-hydroxylase, an enzyme that plays an important role in the detoxification of the cholestatic secondary bile acid lithocholate. Western-blot analysis revealed that the expression of CYP 3A10 protein is male-specific in hamster liver microsomes, a finding that is consistent with earlier analysis of CYP 3A10 mRNA. Since it has not been established whether the specificities of bile acid hydroxylase P450s, such as CYP 3A10, are restricted to their anionic bile acid substrates, we investigated the role of CYP 3A10 in the metabolism of a series of neutral steroid hormones using cDNA directed-expression in COS cells. The steroid hormones examined, testosterone, androstenedione and progesterone, were each metabolized by the expressed CYP 3A10, with 6 beta-hydroxylation corresponding to a major activity in all three instances. CYP 3A10-dependent steroid hydroxylation was increased substantially when the microsomes were prepared from COS cells co-transfected with NADPH:P450 reductase cDNA. In this case, the expressed P450 actively catalysed the 6 beta-hydroxylation of testosterone (288 +/- 23 pmol of product formed/min per mg of COS-cell microsomal protein), androstenedione (107 +/- 19 pmol/min per mg) and progesterone (150 +/- 7 pmol/min per mg). Other major CYP 3A10-mediated steroid hydroxylase activities included androstenedione 16 alpha-hydroxylation, progesterone 16 alpha- and 21-hydroxylation, and the formation of several unidentified products. CYP 3A10 exhibited similar Vmax. values for the 6 beta-hydroxylation of androstenedione and lithocholic acid (132 and 164 pmol/min per mg respectively), but metabolized the bile acid with a 3-fold lower Km (25 microM, as against 75 microM for androstenedione). Together, these studies establish that the substrate specificity of the bile acid hydroxylase CYP 3A10 is not restricted to bile acids, and further suggest that CYP 3A10 can play a physiologically important role in the metabolism of two classes of endogenous P450 substrates:steroid hormones and bile acids.
细胞色素P450 3A10（CYP 3A10）是一种仓鼠肝脏细胞色素P-450（P450），它编码石胆酸6β-羟化酶，该酶在胆汁淤积性次级胆汁酸石胆酸盐的解毒过程中发挥重要作用。蛋白质免疫印迹分析显示，CYP 3A10蛋白在仓鼠肝脏微粒体中的表达具有雄性特异性，这一发现与早期对CYP 3A10 mRNA的分析结果一致。由于尚未确定胆汁酸羟化酶P450（如CYP 3A10）的底物特异性是否仅限于其阴离子胆汁酸底物，我们利用在COS细胞中的cDNA定向表达研究了CYP 3A10在一系列中性甾体激素代谢中的作用。所检测的甾体激素睾酮、雄烯二酮和孕酮均被表达的CYP 3A10代谢，在所有三种情况下，6β-羟化均为主要活性。当从与NADPH：P450还原酶cDNA共转染的COS细胞中制备微粒体时，CYP 3A10依赖性甾体羟化作用显著增强。在这种情况下，表达的P450可有效催化睾酮（每毫克COS细胞微粒体蛋白每分钟形成288±23皮摩尔产物）、雄烯二酮（每毫克107±19皮摩尔/分钟）和孕酮（每毫克150±7皮摩尔/分钟）的6β-羟化。其他主要的CYP 3A10介导的甾体羟化酶活性包括雄烯二酮16α-羟化、孕酮16α-和21-羟化，以及几种未鉴定产物的形成。CYP 3A10对雄烯二酮和石胆酸的6β-羟化表现出相似的最大反应速度值（分别为每毫克132和164皮摩尔/分钟），但代谢胆汁酸时的米氏常数低3倍（25微摩尔，而雄烯二酮为75微摩尔）。总之，这些研究表明胆汁酸羟化酶CYP 3A10的底物特异性并不局限于胆汁酸，进一步表明CYP 3A10在两类内源性P450底物（甾体激素和胆汁酸）的代谢中可能发挥重要的生理作用。
