Localization of the active center of nitroxide radical reduction in rat liver microsomes: its relation to cytochrome P-450 and membrane fluidity.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Utsumi
H
H
Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo.
Shimakura
A
A
Kashiwagi
M
M
Hamada
A
A
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004790:Enzyme Induction / Q000187:drug effects; D066298:In Vitro Techniques; D008297:Male; D008560:Membrane Fluidity; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology*; D009589:Nitrogen Oxides / Q000378:metabolism*; D010084:Oxidation-Reduction; D010634:Phenobarbital / Q000494:pharmacology; D011078:Polychlorinated Biphenyls / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D009589:Nitrogen Oxides; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D011078:Polychlorinated Biphenyls; C039900:nitroxyl; D010634:Phenobarbital