REGULATION AND FUNCTION OF P450III GENE FAMILY ISOZYMES

这项研究的总体目标是阐明其功能和调节 由类固醇诱导的细胞色素P450家族编码的蛋白质 (细胞色素P450III基因)，存在于人类和其他物种中。 尽管这个家庭的个别成员对 底物的代谢尚不清楚，以前的研究表明 这个细胞色素P450家族在细胞色素P450的代谢中起着关键作用。 某些内生菌药物(类固醇)、临床重要药物(环孢素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)。尽管它们之间的氨基酸序列同源性很高 该基因家族的成员(例如，两个基因的氨基酸同源性为89% 同工酶、增殖细胞核抗原和PCNb)，我们认为有可能产生 针对该家族个别成员的单抗。它 区分这些同工酶是很重要的，因为我们假设 它们之间有足够的结构差异，以至于明显 影响代谢的底物专一性和区域选择性 这些同工酶。这些同工酶之间的差异在类型 肝外代谢产物的形成与肝外的差异 分布可能影响致癌物的靶器官特异性和 毒药。该项目的具体目标是：1)生产和 纯化一组特定的、不同表位的和抑制性的单抗 抗大鼠增殖细胞核抗原、PCNb和PCNC抗体部分呈阳性反应 Western blotts，2)用这些抗体评价PCNC 是类固醇诱导的P450家族中的第三个成员 差示免疫亲和层析分离该新成员 产生同工酶特异性单抗的基因家族，4) 克隆此家族的新成员以获取测序信息和 构建特异性探针，5)利用这些抗体进行定量 肝脏和肝脏微粒体的个体同工酶水平 肝外组织，从而提供了对调节 这些蛋白质，6)使用这些抗体来评估 不同个体同工酶对中华绒螯蟹微体代谢的影响 底物，7)以确定人类蛋白质(HLP、HLp2和 细胞色素P450III基因家族的HLp3)具有结构性和/或 大鼠的功能同源物，8)检查选定的猴子组织 P450III家族同源基因的表达样本。影响的因素 外来化合物的活化，包括这些化合物的诱导 代谢多种化合物的酶直接影响人类健康 以及外来生物暴露的风险。

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.