REGULATION AND FUNCTION OF P450III GENE FAMILY ISOZYMES

本研究的总体目标是阐明其功能和调控 类固醇诱导细胞色素P450家族编码的蛋白质 （细胞色素P450 III基因），发现于人类和其他物种。 虽然这个家庭的个别成员对家庭的贡献， 底物的代谢尚不清楚，以前的研究表明， 该细胞色素P450家族在以下代谢中起关键作用： 某些内生菌（类固醇），临床上重要的药物（环孢菌素A 和大环内酯类抗生素），以及广泛存在的环境污染物 （黄曲霉毒素B1）。 尽管氨基酸序列同源性很高， 该基因家族的成员（例如，89%的氨基酸同源性， 同工酶，PCNa和PCNb），我们建议，这是可能的生产 对该家族的单个成员特异的单克隆抗体。 它 区分这些同工酶很重要，因为我们假设， 它们之间存在足够的结构差异， 影响代谢的底物特异性和区域选择性， 这些同工酶。 这些同工酶的类型之间的差异 代谢物形成的组合与差异，在肝外 分布可能影响致癌物的靶器官特异性， 有毒物质 该项目的具体目标是：1）生产和 纯化一组特异性、表位特异性和同源性单克隆抗体， 抗大鼠PCNa、PCNb和PCNc的抗体，其中一些在 蛋白质印迹，2）使用这些抗体来评估PCNc是否 是类固醇诱导型P450家族的第三个成员，3）使用 差异免疫亲和层析分离新成员， 用于产生同工酶特异性单克隆抗体的基因家族，4） 克隆该家族的新成员以获得测序信息， 特异性探针的构建，5）使用这些抗体来定量 肝脏微粒体中单个同工酶的水平， 肝外组织，从而提供了一个了解的调节， 这些蛋白质，6）使用这些抗体来评估的贡献， 个别同工酶对微粒体代谢的选择 底物，7）以确定人类蛋白质（HLp、HLp 2和HLp 3）中的哪一种是人源蛋白质（HLp、HLp 2和HLp 3）。 细胞色素P450 III基因家族的HLp 3）是结构性的和/或 大鼠的功能同源物，8）检查选定的猴组织 用于表达P450 III家族同源物的样品。 的影响因素 激活外来化合物，包括诱导这些 代谢多种化合物的酶直接影响人体健康 以及暴露在异生物质中的风险。

The overall aim of this research is to clarify the functions and regulation of the proteins coded by the steroid-inducible cytochrome P450 family (cytochrome P450III gene) which is found in humans and other species. Although the contributions of individual members of this family toward the metabolism of substrates are unclear, previous studies have indicated that this family of cytochrome P450 plays a pivotal role in the metabolism of certain endobiotics (steroids), clinically important drugs (cyclosporin A and macrolide antibiotics), and widely occurring environmental contaminants (aflatoxin B1). Despite the high amino acid sequence homology among members of this gene family (e.g., 89% amino acid homology for two isozymes, PCNa and PCNb) we propose that it is possible to produce monoclonal antibodies specific for individual members of this family. It is important to distinguish between these isozymes as we hypothesize that there are sufficient structural differences between them to markedly influence the substrate specificity and regioselectivity of metabolism by these isozymes. These differences between isozymes in the types of metabolites formed in combination with differences in the extrahepatic distribution may influence the target organ specificity of carcinogens and toxicants. The specific aims of this project are: 1) to produce and purify a battery of specific-, epitope distinct- and inhibitory-monoclonal antibodies against rat PCNa, PCNb and PCNc some of which are positive on Western blots, 2) to use these antibodies to evaluate whether or not PCNc is a third member of the steroid-inducible family of P450, 3) to use differential immunoaffinity chromatography to isolate new members of this gene family for production of isozyme specific monoclonal antibodies, 4) to clone new members of this family for sequencing information and construction of specific probes, 5) to use these antibodies to quantitate the levels of individual isozymes in microsomes from the liver and extrahepatic tissues and thus provide an understanding of the regulation of these proteins, 6) to use these antibodies to assess the contributions of individual isozymes toward the microsomal metabolism of selected substrates, 7) to determine which of the human proteins (HLp, HLp2 and HLp3) of the cytochrome P450III gene family are structural and/or functional homologs of the rat, 8) to examine selected monkey tissue samples for expression of P450III family homologs. Factors which influence the activation of foreign compounds, including the induction of these enzymes which metabolize many compounds, directly influence human health and the risk from xenobiotic exposure.