HUMAN LIVER CYTOCHROMES P450

我们建议继续对人类P450酶进行研究，这些酶参与了 外源物质的代谢，重点放在这些蛋白质上 包括CYP2C基因亚家族，即P4502C8、P4502C9、P4502C18 和P4502C19。在这一过程中，CYP2C酶具有特别重要的作用 或更多的这些蛋白质可能是氧化的几种多态的基础 人类的药物新陈代谢。因此，我们的计划包括特定的CYP2C 参与S-美苯妥因羟化的酶(S-甲苯妥英钠)和 甲苯丁胺(TOL)，两种治疗药物，表现出明显的 个体间在肝脏代谢方面的差异。 将首先确定2C8、2C9、2C18和2C19的参与程度 用从人类肝脏中提纯的这些酶重组的系统。 后续研究将使用具有抑制作用的完整肝微体 适当的CYP2C酶的抗体(S)，以证实 用纯化的重组蛋白获得的结果。另一位 我们的目标是鉴定表现出异常的人类细胞色素P450 2 C蛋白 功能属性，即负责S-Meph和TOL的属性 代谢多态。为此，我们将首先评估 单个人肝样本氧化S-甲氧基苯丙氨酸和三苯氧胺的能力。 以抗体为基础的CYP2C酶的定量以前的特征是 主要的S-甲氧基苯丙氨酸和TOL羟基酶将允许鉴定 具有CYP2C蛋白的受试者，免疫反应正常但较差 代谢活动。经过纯化后，这些CYP2C酶将 要经过详细的物理特征，包括氨基酸 对蛋白酶衍生的多肽进行序列分析，以确定其特异性 引起蛋白质一级结构改变的蛋白质 功能。在P450酶的表达方面，我们的主要目标是 首先确定肝脏中是否有任何CYP2C蛋白可被诱导 外来生物(如苯巴比妥和利福平)。对于这些研究，我们 将使用人类肝细胞，这是一种成熟的体外系统，允许 操纵细胞世界，但排除了伦理 活体人体实验固有的考虑因素。自.以来 肝细胞将来自主要由采购提供的肝脏。 这些调查还可以揭示这些机构是否适合 研究P450功能和调节的材料，一个问题 学术和工业科学家的重要性。我们的总体目标 是为了确定CYP2C基因亚家族酶在肝药中的作用 新陈代谢，并最终描述了 个体间的变异在它们的功能中被注意到。

We propose to continue our studies on human P450 enzymes involved in the metabolism of xenobiotics, with major emphasis placed on those proteins comprising the CYP2C gene subfamily, namely P4502C8, P4502C9, P4502C18, and P4502C19. The CYP2C enzymes are of particular relevance in that one or more of these proteins may underlie several polymorphisms of oxidative drug metabolism in man. Thus, our plan includes the specific CYP2C enzymes involved in the hydroxylation of S-mephenytoin (S-MEPH) and tolbutamide (TOL), two therapeutic agents that exhibit marked interindividual differences with regards to their hepatic metabolism. The involvement of 2C8, 2C9, 2C18, and 2C19 will first be determined in systems reconstituted with these enzymes purified from human liver. Subsequent studies will employ intact liver microsomes with inhibitory antibodies to the appropriate CYP2C enzyme(s) in order to corroborate the results obtained with the purified reconstituted proteins. Another of our aims is to identify human CYP2C proteins that exhibit aberrant functional properties, i.e., those responsible for the S-MEPH and TOL metabolic polymorphisms. For this purpose, we will first assess the capacity of individual human liver samples to oxidize S-MEPH and TOL. Antibody-based quantitation of CYP2C enzymes previously characterized as the major S-MEPH and TOL hydroxylases will allow identification of subjects who possess CYP2C proteins with normal immunoreactivity yet poor metabolic activity. After their purification, these CYP2C enzymes will be subjected to detailed physical characterization, including amino acid sequence analysis of protease-derived peptides, to ascertain the specific changes in protein primary structure that give rise to alterations in function. In terms of P450 enzyme expression, our primary goal is to first resolve whether any of the liver CYP2C proteins are inducible by xenobiotics (e.g., phenobarbital and rifampicin). For these studies, we will employ human hepatocytes, an established in vitro system allowing for manipulation of the cellular mileau but precluding the ethical considerations inherent to in vivo human experimentation. Since hepatocytes will be derived from livers provided mainly by procurement agencies, these investigations can also reveal the suitability of such material for studying P450 function and regulation, a question of importance of both academic and industrial scientists. Our overall goals are to define a role for the CYP2C gene subfamily enzymes in hepatic drug metabolism and, ultimately, to describe a biochemical basis for the interindividual variations noted in their function.