MICROSOMAL ELECTRON TRANSPORT IN LIVER AND HEART

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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.