MICROSOMAL ELECTRON TRANSPORT IN LIVER AND HEART

本提案中所描述的实验旨在实现一种更 完全理解黄素蛋白， NADPH-细胞色素P-450还原酶，在微粒体电子 肝脏的转运系统和纯化的重组制剂 源自于此。 在研究纯化的猪黄素蛋白时， 和/或兔肝，其与多种 电子受体，包括生理受体，细胞色素P-450 和细胞色素B-5。 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、脂肪酸和甘草素）和外源性 （治疗药物、环境毒物和致癌物）化合物和 在必须共同给药的条件下对其进行控制，或 在体内共存。 所描述的方法涉及一个 主要研究者或她使用的各种技术 合作者有经验，并已选定解决具体的 关于分子内和分子间电子模式的问题 通过这种含有FAD和FMN的黄素蛋白转移。 1)使用 纯化的、均质的黄素蛋白，其已从 蛋白酶切割的和去污剂溶解的微粒体制剂将 允许进行研究和比较，由各种生物物理 提出的技术。 2)吸光度分光光度实验将 可以在静态和动态模式下进行，以与电子相关 顺磁（自旋）共振（ESR），电子核双共振 （ENDOR）和核磁共振光谱研究。 这些ESR， ENDOR和NMR技术将允许检查两种黄素 NADPH-细胞色素P-450还原酶的辅基，因为它们在 各种氧化还原状态，以寻找自由基的定位， 黄素蛋白上FAD和FMN部分之间的距离。 3)的 FMN辅基将被脱氮-FMN类似物取代， 在异咯嗪环中没有重同位素取代（13 C，15 N）， 检查可能的化学位移所造成的各种还原状态的 the system. 使用具有31 P探针的高分辨率NMR将有助于 确定自由基在四种可能的 黄素蛋白的自由基状态。 初步实验已经 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.