CYTOCHROME P-450 MULTIPLICITY AND FUNCTION

CYTOCHROME P-450 的多样性和功能

• 批准号: 3250860
• 负责人: LAURENCE S KAMINSKY
• 金额: $ 7.41万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1987-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3250860
• 关键词: NAD(P)H dehydrogenase; antibody specificity; chemical structure function; circular dichroism; colorimetry; conformation; cytochrome P450; density gradient ultracentrifugation; detoxification; drug metabolism; electron microscopy; environmental contamination; environmental toxicology; enzyme structure; high performance liquid chromatography; human tissue; immunoelectrophoresis; isozymes; lipids; microsomes; noninvasive diagnosis; species difference; spectrometry; stereochemistry; toxin metabolism; ultracentrifugation; warfarin

The hepatic mixed-function oxidase system with cytochrome P-450 (P-450) as the terminal oxidase plays a major role in detoxifying and toxifying a wide variety of drugs and environmental pollutants, and in hydroxylating endogenous substrates such as steroids and fatty acids. It plays a key role in chemical carcinogenesis, pharmacology, and toxicology. The long term objective of these studies is to understand the function and control of P-450, to utilize its beneficial capabilities and prevent its hazardous activity. The specific aims are to determine the basis for functional differences between P-450 isozymes, particularly the role of the microsomal milieu and isozyme conformation, and the multiplicity of P-450s in animals and humans. The overall approach is to use a novel, well developed analytical method, where the metabolism of the R and S enantiomers of the anticoagulant drug warfarin to multiple metabolites, together with the inhibition of this metabolism by antibodies against specific P-450 isozymes, will be used as a probe of P-450 isozyme function and composition. Functional studies will be performed on five highly purified rat liver P-450 isozymes. Specific aspects of the role of the microsomal environment which will be studied are the role of lipid vesicles, cytochrome b5, NADPH-cytochrome P-450 reductase, and mixtures of P-450 isozymes. Conformational modifications will be mediated by detergents, urea, thermally and by chemical modification of amino acid residues, and will be monitored by circular dichroism and differential scanning microcalorimetry. Effects of conformational changes on function will be monitored by warfarin metabolism. Multiplicity will be studied in monkey, guinea pig, pig and human liver and compared with previous results with rats, rabbits, and mice. Multiplicity of P-450 will be assessed by comparing P-450 isozyme compositions in these animals and in man based on the regio- and stereoselectivity of warfarin metabolism, its specific inhibition by antibodies to P-450 isozymes, and by immunoelectrophoretic methods. Interindividual differences between human P-450 isozymes will be assessed. Two major benefits related to human health will be the development of a unique noninvasive method for determining human liver P-450 isozyme compositions, and the development of the basis for evaluating extrapolations of animal toxicology data from animals to humans.

以细胞色素P-450（P-450）为底物的肝混合功能氧化酶系统， 末端氧化酶在广泛的解毒和解毒过程中起着重要作用。 各种药物和环境污染物，并在羟基化 内源性底物如类固醇和脂肪酸。 起着关键 在化学致癌作用、药理学和毒理学中的作用。 长 这些研究的长期目标是了解功能和控制 P-450，以利用其有益的能力，并防止其危险 活动 具体目标是确定函数的基础 P-450同工酶之间的差异，特别是微粒体的作用 环境和同工酶构象，以及动物中P-450的多样性 还有人类 总体方法是使用一种新颖的、开发良好的 分析方法，其中代谢的R和S对映异构体的 抗凝药物华法林的多种代谢产物，连同 通过抗特异性P-450的抗体抑制这种代谢 同工酶，将用作P-450同工酶功能的探针， 混合物. 将对5个高度纯化的 大鼠肝P-450同工酶。 微粒体作用的具体方面 将被研究的环境是脂质囊泡的作用， 细胞色素b5、NADPH-细胞色素P-450还原酶和P-450的混合物 同工酶 构象修饰将由去污剂介导， 尿素，热修饰和化学修饰氨基酸残基，和 将通过圆二色性和差示扫描进行监测 微量热法 构象变化对功能的影响将是 通过华法林代谢监测。 将在猴中研究多重性， 豚鼠，猪和人的肝脏，并与以前的结果进行比较， 大鼠、兔子和小鼠。 P-450的多重性将通过以下方式进行评估： 比较这些动物和人类的P-450同工酶组成， 华法林代谢区域和立体选择性，其特异性 P-450同工酶抗体抑制和免疫电泳 方法. 人类P-450同工酶之间的个体差异将在 评估。 与人类健康相关的两个主要益处是 一种独特的非侵入性方法的开发，用于确定人类肝脏 P-450同工酶组成及其评价基础的建立 从动物到人类的动物毒理学数据外推。

项目成果

Human hepatic cytochrome P-450 composition as probed by in vitro microsomal metabolism of warfarin.

• 发表时间: 1984-07
• 期刊: Drug metabolism and disposition: the biological fate of chemicals
• 作者: L. Kaminsky;D. Dunbar;P. Wang;P. Beaune;D. Larrey;F. Guengerich;R. Schnellmann;I. Sipes