MICROSOMAL ELECTRON TRANSPORT IN LIVER AND HEART

这项提案中描述的实验旨在实现更多 完全理解黄素蛋白， NADPH-细胞色素P-450还原酶在微粒体电子中的作用 肝脏转运系统和纯化、重组制剂中的 从那里衍生出来的。提纯猪黄素蛋白的研究 和/或兔肝，其与多种物质相互作用的机制 电子受体，包括生理受体细胞色素P-450 并对细胞色素b-5进行检测。对这些分子的理解 机制是预测两者相互作用的能力的基础 内源性(类固醇、脂肪酸和前列腺素)和外源性 (治疗药物、环境毒物和致癌物质)化合物和 在必须共同管理或共同管理的条件下进行控制 否则就会在身体里共存。所描述的方法涉及一个 首席调查员或她所使用的各种技术 协作者有经验，并已被选中解决特定问题 关于分子内和分子间电子模式的几个问题 通过这种含有FAD和FMN的黄素蛋白进行转移。1)使用 纯化的、均一的黄素蛋白已经从两者中制备 蛋白酶裂解和洗涤剂增溶的微粒体制剂将 允许由不同的生物物理学家进行研究和比较 建议的技术。2)分光光度实验将 在静态和动态模式下执行，以便与电子关联 顺磁(自旋)共振(ESR)、电子核双共振 (Endor)和核磁共振波谱研究。这些ESR， Endor和核磁共振技术将允许检查两个Flavin NADPH-细胞色素P-450还原酶的辅基在相互作用中的作用 各种氧化还原状态以寻找自由基和 黄素蛋白上FAD和FMN部分之间的距离。3) FMN假体基团将被Daza-FMN类似物取代，并 在异四氧氮环上没有重同位素取代(13C，15N)到 研究不同还原状态可能导致的化学位移 这个系统。使用31P探头的高分辨率核磁共振将有助于 自由基在四种可能中位置的确定 黄素蛋白的自由基态。初步实验已经完成 已执行。

The experiments described in this proposal are directed toward a more complete understanding of the mechanism by which the flavoprotein, NADPH-cytochrome P-450 reductase, functions in the microsomal electron transport system of liver and in purified, reconstituted preparations derived therefrom. In studying the purified flavoprotein from porcine and/or rabbit liver, the mechanism of its interaction with a variety of electron acceptors, including the physiological acceptors, cytochrome P-450 and cytochrome b-5 will be examined. The understanding of these molecular mechanisms underlies the ability to predict the interactions of both endogenous (steroids, fatty acids, and prostaglandins) and exogenous (therapeutic drugs, environmental toxicants, and carcinogens) compounds and their control under conditions in which they must be co-administered or otherwise co-exist in the body. The methodology described involves a variety of techniques with which the Principal Investigator or her collaborators have experience and has been selected to address specific questions about the mode of both intramolecular and intermolecular electron transfer by this FAD- and FMN-containing flavoprotein. 1) The use of purified, homogeneous flavoprotein which has been prepared from both protease-cleaved and detergent-solubilized microsomal preparations will permit studies to be performed and compared by the various biophysical techniques proposed. 2) The absorbance spectrophotometric experiments will be performed in static and kinetic modes for correlation with the electron paramagnetic (spin) resonance (ESR), electron nuclear double resonance (ENDOR), and nuclear magnetic resonance spectrometric studies. These ESR, ENDOR, and NMR techniques will permit the examination of the two flavin prosthetic groups of NADPH-cytochrome P-450 reductase as they interact in various redox states in search of the localization of the free radical and the distances between the FAD and FMN moieties on the flavoprotein. 3) The FMN prosthetic group will be substituted with deaza-FMN analogs with and without heavy isotope substitutions (13C, 15N) in the isoalloxazine ring to examine possible chemical shifts resulting from various reduction states of the system. The use of high resolution NMR with a 31P probe will aid in the determination of the location of the free radical in the four possible free radical states of the flavoprotein. Preliminary experiments have been performed.