ASSESSMENT OF HUMAN P-450 CATALYTIC ACTIVITIES BY CDNA-DIRECTED EXPRESSION

P-450是一组血红素蛋白，是终末组分 混合功能单加氧酶系统。 这些酶 一个基因超家族的产物和多达50种不同的形式， 存在于哺乳动物中。 大部分P-450在肝脏中表达为 负责代谢外来化学物质的酶。 在 在某些情况下，内源性类固醇激素与 不同种类的P-450;然而， 这些反应值得怀疑。 最重要的是 P-450参与化学致癌物活化。 的 研究P-450酶学的一般方法是通过标准 酶的纯化和测定。 这有两个主要缺点 方法是难以获得高度纯化的 制备和事实，这些膜结合酶必须 用人造脂质双层和酶重构 NADPH-P-450-氧化还原酶。 人类P-450甚至更多 由于缺乏可用的组织而难以纯化 源头 我们一直在研究大鼠和人类P- 450 s。 几种P450已经使用多种表达方法表达， 系统. 大鼠IIAI、IIDI和IVAI以及人IIC 8、IIC 9、IIDI和IVAI IIAI P450已经使用COS细胞系统表达。 鼠标 IAI和IA 2，以及大鼠IIAI和IVAI使用T7- 牛痘病毒系统。 大鼠IIAI和IVAI使用 酵母表达系统 这些酶和其他酶也被 使用杆状病毒和逆转录病毒表达。 表达的P-450已被评价其催化活性 各种药物和致癌物的反应 DNA结合和 诱变试验被用来评估致癌物质 人类P-450的激活特性

P-450s are a group of hemoproteins that are the terminal components of the mixed function monooxygenase system. These enzymes are products of a gene superfamily and up to 50 distinct forms may exist in mammals. The bulk of P-450s are expressed in liver as enzymes responsible for the metabolism of foreign chemicals. In some cases endogenous steroid catabolism is associated with distinct species of P-450s; however, the physiological role of these reactions is questionable. Of principal importance is the participation of P-450s in chemical carcinogen activation. The general method for study of P-450 enzymology is through standard purification and assay of the enzymes. Two major drawbacks to this approach are the difficulty in obtaining highly purified preparations and the fact that these membrane-bound enzymes must be reconstituted with artificial lipid bilayers and the enzyme NADPH-P-450-oxidoreductase. Further human P-450s are even more difficult to purify based on the paucity of an available tissue source. We have been studying the enzymology of rat and human P- 450s by using cDNA-directed expression. Several P450s have been expressed using a variety of expression systems. Rat IIAI, IIDI and IVAI and human IIC8, IIC9, IIDI and IIAI P450s have been expressed using the COS-cell system. Mouse IAI and IA2, and rat IIAI and IVAI were expressed using the T7- vaccinia virus system. Rat IIAI and IVAI were expressed using a yeast expression system. These enzymes and others are also being expressed using baculo virus and retrovirus. Expressed P-450s have been evaluated for their catalytic activities toward a variety of drugs and carcinogens. DNA binding and mutagenesis assays are being used to evaluate the carcinogen activating properties of human P-450s.