IDENTIFICATION AND CHARACTERIZATION OF NEW HUMAN P-450

细胞色素P450是外源化合物代谢的主要酶， 氧化和分解大多数临床使用的药物， 更容易从体内排出。 P450也能激活化学物质 致癌物转化为能够与DNA结合的高活性亲电体 和变异基因 P450存在很大程度的物种差异 基因，特别是啮齿动物和人类之间。 这些差异在 调节和催化水平，并且在基因之间最普遍 在不同的CYP2亚家族中。 CYP2A、CYP2B和CYP2C亚家族 特别复杂，成员众多。cDNA编码两种不同的 人CYP2A和CYP2B P450以及四种CYP2C P450已在 人类 由于这些亚科中的许多啮齿动物P450 已经确定，存在多种人CYP2A、CYP2B和 Southern印迹分析提示CYP2C基因，我们认为， 这些亚家族中的其他人P450还有待鉴定。 我们 因此，开发了使用聚合酶链反应（PCR）的策略， 找到并分离出新的人类P450 用PCR方法鉴定CYP2C18， 克隆cDNA并进行全序列测定。 将cDNA表达为 相对分子量为49，000的蛋白质。 我们没有， 然而，鉴定了该酶的有利底物。 基于pcr的 开发了一种测定方法，用于定量CYP2C8、CYP2C9和CYP2CI8的水平 mRNA。 研究正在进行中，以确定人类的催化特异性。 使用cDNA表达的P450。 抗凝剂华法林的代谢 使用牛痘病毒表达的P450研究。 代谢 致癌物致突变代谢物进行了分析，通过使用 淋巴母细胞样cDNA表达系统。

Cytochrome P450s, the principal enzymes of foreign compound metabolisms, oxidize and inactivate most of the clinically used drugs so that they can be more easily eliminated from the body. P450s also activate chemical carcinogens to highly reactive electrophiles capable of binding to DNA and mutating genes. A large degree of species differences exist for P450 genes, particularly between rodents and humans. These differences are at the regulatory and catalytic level and are most prevalent between genes in the various CYP2 subfamilies. The CYP2A, CYP2B and CYP2C subfamilies are particularly complex with many members. cDNAs encoding two different human CYP2A and CYP2B P450s and four CYP2C P450s have been identified in humans. Since numerous rodent P450s within each of these subfamilies have been identified and the presence of multiple human CYP2A, CYP2B and CYP2C genes are suggested by Southern blotting analysis, we believe that other human P450s within these subfamilies have yet to be identified. We therefore developed strategies using polymerase chain reactions (PCR) to find and isolate new human P450s. CYP2CI8 was identified by PCR and its CDNA was cloned and completely sequenced. The CDNA was expressed into a protein with a relative molecular weight of 49,000. We have not, however, identified a favorable substrate for this enzyme. A PCR-based assay was developed to quantify levels of CYP2C8, CYP2C9 and CYP2CI8 MRNA. Studies are in progress to determine the catalytic specificities of human P450s using CDNA expression. Metabolism of the anticoagulant warfarin was studied using vaccinia virus-expressed P450s. Metabolism of carcinogens to mutagenic metabolites was analyzed by use of the lymphoblastoid CDNA expression system.