Localization of the Active Center of Nitroxide Radical Reduction in Rat Liver Microsomes: Its Relation to Cytochrome P-450 and Membrane Fluidity1

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Abstract
The properties and localization of the active center of NADPH-dependent nitroxide radical reduction in rat liver microsomes were investigated with the following five spin-probes as substrates; tetramethylpiperidinol-N-oxyl (TEMPOL) and four spin-labeled stearic acid derivatives with a nitroxide radical at the 5th, 7th, 12th, or 16th position of the hydrocarbon chain (abbreviated as 5SLS, 7SLS, 12SLS, and 16SLS, respectively). The ESR signals of these spin-probes in microsomes decreased on the addition of NADPH, and the decay was inhibited by pretreatment with SKF-525A. Experiments with various microsomal preparations induced by phenobarbital(PB), polychlorinated biphenyls (PCB), or 3-methylcholanthrene (3-MC) revealed that the reduction rate was correlated to the concentration of cytochrome P-450 but not to that of NADPH reductase. Thus, the nitroxide radicals of the SLSs and TEMPOL seem to be reduced by the combined action of NADPH-cytochrome P-450 reductase and cytochrome P-450. The decay showed a lag time, but no distinct correlation was observed between the lag time and the spin-probe species. On the other hand, the initial velocity of the nitroxide reduction depended strongly on the spin-probe species. Among the five spin-probes, 7SLS was reduced most quickly, followed by 5SLS, 12SLS, TEMPOL, and 16SLS in that order. The reduction rate varied from 0.18/min for 7SLS to 0.08/min for 16SLS. There was a linear relation between the cytochrome P-450 content and the reduction rate. The specific activity of cytochrome P-450 for the reduction of each SLS showed that the active center of the nitroxide radical reduction, in which cytochrome P-450 is a major component, should be located at about the depth of the 7th position of stearic acid in the membranes. The membrane fluidity of microsomes was estimated at the same time from the spectra of the four SLSs, but no significant correlation was observed between the nitroxide reduction and the membrane fluidity of liver microsomes.