Waxman D J, Lapenson D P, Aoyama T, Gelboin H V, Gonzalez F J, Korzekwa K
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
Arch Biochem Biophys. 1991 Oct;290(1):160-6. doi: 10.1016/0003-9861(91)90602-f.
Steroid hydroxylation specificities were determined for 11 forms of human cytochrome P450, representing four gene families and eight subfamilies, that were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. Microsomes isolated from the P450-expressing Hep G2 cells were isolated and then assayed for their regioselectivity of hydroxylation toward testosterone, androstenedione, and progesterone. Four of the eleven P450s exhibited high steroid hydroxylase activity (150-900 pmol hydroxysteroid/min/mg Hep G2 microsomal protein), one was moderately active (30-50 pmol/min/mg) and six were inactive. In contrast, 10 of the P450s effectively catalyzed O-deethylation of 7-ethoxycoumarin, a model drug substrate, while only one (P450 2A6) catalyzed significant coumarin 7-hydroxylation. Human P450 4B1, which is expressed in lung but not liver, catalyzed the 6 beta-hydroxylation of all three steroids at similar rates and with only minor formation of other hydroxylated products. Three members of human P450 family 3A, which are expressed in liver and other tissues, also catalyzed steroid 6 beta-hydroxylation as their major activity but, additionally, formed several minor products that include 2 beta-hydroxy and 15 beta-hydroxy derivatives in the case of testosterone. These patterns are similar to those exhibited by rat family 3A P450s. Although several rodent P450s belonging to subfamilies 2A, 2B, 2C, 2D are active steroid hydroxylases, four of five human P450s belonging to these subfamilies exhibited very low activity or were inactive, as were the human 1A and 2E P450s examined in the present study. These studies demonstrate that individual human cytochrome P450 enzymes can hydroxylate endogenous steroid hormones with a high degree of stereospecificity and regioselectivity, and that some, but not all of the human cytochromes exhibit metabolite profiles similar to their rodent counterparts.
利用痘苗病毒通过cDNA定向表达，在人肝癌Hep G2细胞中合成了代表四个基因家族和八个亚家族的11种人细胞色素P450，测定了它们的类固醇羟化特异性。从表达P450的Hep G2细胞中分离出微粒体，然后检测其对睾酮、雄烯二酮和孕酮的羟化区域选择性。11种P450中有4种表现出高类固醇羟化酶活性（150 - 900 pmol羟类固醇/分钟/毫克Hep G2微粒体蛋白），一种活性中等（30 - 50 pmol/分钟/毫克），六种无活性。相比之下，10种P450能有效催化模型药物底物7 - 乙氧基香豆素的O - 脱乙基反应，而只有一种（P450 2A6）催化显著的香豆素7 - 羟化反应。在肺而非肝脏中表达的人P450 4B1以相似的速率催化所有三种类固醇的6β - 羟化反应，且仅少量生成其他羟化产物。在肝脏和其他组织中表达的人细胞色素P450家族3A的三个成员也以类固醇6β - 羟化作为主要活性，但此外，在睾酮的情况下还生成了几种次要产物，包括2β - 羟基和15β - 羟基衍生物。这些模式与大鼠家族3A P450所表现出的模式相似。尽管属于2A、2B、2C、2D亚家族的几种啮齿动物P450是活性类固醇羟化酶，但属于这些亚家族的五种人P450中有四种表现出非常低的活性或无活性，本研究中检测的人1A和2E P450也是如此。这些研究表明，个体人细胞色素P450酶能够以高度的立体特异性和区域选择性对内源性类固醇激素进行羟化，并且一些（但不是全部）人细胞色素表现出与其啮齿动物对应物相似的代谢产物谱。
