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STRUCTURE, FUNCTION, & DYNAMICS OF P-450 CYTOCHROMES

结构、功能、

基本信息

批准号：
3466231
负责人：
SHAUN BLACK
金额：
$ 6.71万
依托单位：
UNIVERSITY OF TEXAS HLTH CTR AT TYLER
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-02-01 至 1994-01-31
项目状态：
已结题

项目摘要

Structure, function, and conformational dynamics of the cytochrome P-450 monooxygenase system are to be studied in the proposed research. (1) Topology of membrane-bound rabbit microsomal isozymes 2 and 4 will be probed via polyclonal and monoclonal antibodies as well as by specific and non-specific proteolysis. Immobilized polyclonal antibodies will be used to purify specific peptides released upon protease treatment of microsomes; HPLC-isolated peptides will be submitted to micro- sequence analysis to reveal sites within the primary structures available at the external (cytoplasmic) surface of the endoplasmic reticulum. A panel of monoclonal antibodies will be raised to synthetic peptides selected from each sequence by MSEQ computer analysis; these site-specific antibodies will be utilized to probe native and protease-digested microsomes via ELISA and Western-blotting procedures. Together, the results obtained will permit reconstruction of the membrane topological features of each cytochrome. Preliminary data and calculations suggest, in contrast to current theory, that the topology of the P-450's is simple and is similar to that known for other microsomal proteins. Isozymes 2 and 4 were chosen for the proposed studies as they are the major forms present in phenobarbital-induced (isozyme 2) and aryl hydrocarbon-induced (isozyme 4) animals, they represent two major gene sub-families, they typify low- and high-spin cytochrome types, they are involved in drug disposition tolerance, and they participate in the oxidative metabolism of physiological lipids, numerous drugs, and other xenobiotics; furthermore, they are highly similar to forms known in the human. (2) Conformational dynamics of isozyme 2 will be studied with site- specific chemical modification of Cys-152, a residue shown to serve as a "reporter" for binding events at the active site. Modification kinetics, carbon-13 NMR spectroscopy, and circular dichroism will be utilized to examine the role and importance of conformational changes in the function of the cytochrome. Functional aspects to be examined will include substrate binding, heme reduction kinetics, and protein-protein interactions. (3) Crystallization of many of the microsomal monooxygenase system components will be attempted with the ultimate aim of three- dimensional structure elucidation. Exploration of the "small Amphiphile" concept and co-crystallization will be major thrusts. A long-term objective of these studies is to achieve a sophisticated molecular view of the P-450 system that will serve to guide rational drug design.

结构、功能和构象动力学细胞色素 P-450 单加氧酶系统的研究提出的研究。 (1)膜结合兔的拓扑结构微粒体同工酶 2 和 4 将通过多克隆和单克隆抗体以及特异性和非特异性抗体蛋白水解。固定化多克隆抗体将用于纯化蛋白酶处理后释放的特定肽微粒体； HPLC 分离的肽将提交给微生物序列分析揭示主要结构内的位点在内质的外（细胞质）表面可用网状结构。将产生一组单克隆抗体通过 MSEQ 从每个序列中选择合成肽计算机分析；将利用这些位点特异性抗体通过 ELISA 探测天然微粒体和蛋白酶消化的微粒体蛋白质印迹程序。总之，所获得的结果将允许重建膜拓扑特征每种细胞色素。初步数据和计算表明，与当前理论相反，P-450 的拓扑结构是简单并且与已知的其他微粒体蛋白相似。同工酶 2 和 4 被选用于拟议的研究，因为它们是苯巴比妥诱导的主要形式（同工酶 2）和芳基碳氢化合物诱导的（同工酶 4）动物，它们代表两种主要基因亚家族，它们代表低自旋和高自旋细胞色素类型，它们涉及药物处置耐受性，它们参与生理的氧化代谢脂质、多种药物和其他外源物质；此外，他们与人类已知的形式高度相似。 (2) 同工酶 2 的构象动力学将通过位点进行研究 Cys-152 的特定化学修饰，该残基显示出充当活性位点结合事件的“报告者”。改性动力学、碳 13 NMR 光谱和圆形将利用二色性来检查的作用和重要性细胞色素功能的构象变化。要检查的功能方面将包括底物结合，血红素还原动力学和蛋白质-蛋白质相互作用。 (3) 许多微粒体单加氧酶系统的结晶将尝试组件的最终目标是三个维度结构阐明。对“小两亲物”概念和共结晶将是主要推动力。这些研究的长期目标是实现 P-450 系统的复杂分子视图将用于指导合理的药物设计。