HUMAN LIVER CYTOCHROMES P450

我们建议继续对参与该过程的人类P450酶进行研究 外源性物质的代谢，主要重点放在这些蛋白质上 包括CYP 2C基因亚家族P4502 C8、P4502 C9、P4502 C18， P4502C19 CYP 2C酶在这一过程中特别重要 这些蛋白质中的一种或多种可能是氧化还原酶的几种多态性的基础。 因此，我们的计划包括特定的CYP 2C 参与S-美芬妥英（S-MEPH）羟基化的酶， 甲苯磺丁脲（TOL），两种治疗剂，表现出显着的 关于他们的肝脏代谢的个体间差异。 2C 8、2C 9、2C 18和2C 19的参与将首先在 用这些从人类肝脏中纯化的酶重建的系统。 随后的研究将使用具有抑制性的完整肝微粒体。 适当CYP 2C酶的抗体，以证实 用纯化的重构蛋白质获得的结果。 中的另一 我们的目的是鉴定出表现出异常CYP 2C蛋白质， 功能特性，即，负责S-MEPH和TOL的人员 代谢多态性 为此，我们将首先评估 单个人肝样品氧化S-MEPH和TOL的能力。 先前表征为CYP 2C酶的基于抗体的定量 主要的S-MEPH和TOL羟化酶将允许鉴定 CYP 2C蛋白免疫反应性正常但较差的受试者 代谢活动 纯化后，这些CYP 2C酶将 进行详细的物理表征，包括氨基酸 蛋白酶衍生肽的序列分析，以确定特异性 蛋白质一级结构的变化， 功能 在P450酶表达方面，我们的主要目标是 首先解决是否有任何肝脏CYP 2C蛋白是可诱导的， 异生物质（例如，苯巴比妥和利福平）。 对于这些研究，我们 将使用人肝细胞，这是一种已建立的体外系统， 但排除了伦理上的 考虑体内人体实验固有的因素。 以来 肝细胞将来自主要通过采购提供的肝脏 这些调查也可以揭示这种机构的适用性， 研究P450功能和调节的材料， 学术和工业科学家的重要性。 我们的总体目标 旨在确定CYP 2C基因亚家族酶在肝脏药物治疗中的作用 代谢，并最终描述了生物化学基础， 在其功能中注意到个体间的差异。

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.