Comparison of highly purified sheep liver and lung NADPH-cytochrome P-450 reductases by the analysis of kinetic and catalytic properties.
1. Reductase was purified to electrophoretic homogeneity from sheep liver and lung microsomes. The specific activity of both enzymes ranged from 55 to 66 mumol cytochrome c reduced/min/mg protein. 2. Liver and lung reductases appeared to have similar kinetic and spectral properties. Km (NADPH) and Km (cytochrome c) values were calculated to be 14.3 +/- 1.23 microM and 22.2 +/- 2.78 microM for liver and 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM for lung reductase, respectively. Kinetic studies showed that cytochrome c can bind the oxidized form of the enzyme as well as its reduced form and both reductases operated through a ping-pong type mechanism. 3. These reductases cannot be distinguished on the basis of monomer molecular weights (Mr 78,000) except that the liver reductase was found to be more susceptible to proteolytic attack. 4. Both reductases supported aniline 4-hydroxylation and ethylmorphine N-demethylation reactions to the same extent in the reconstituted systems. However, sheep lung reductase appeared only 36.5 and 14.8% as effective in catalyzing benzo[a]pyrene reaction as an equivalent amount of reductase from liver in the presence of liver cytochrome P-450 and 3MC-treated rat liver cytochrome P-448, respectively.
Işcan
M Y
MY
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey.
Arinç
E
E
D000818:Animals; D001580:Benzopyrenes / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology*; D009245:NADH Dehydrogenase / Q000302:isolation & purification; D009249:NADP / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification*; D010084:Oxidation-Reduction / Q000187:drug effects; D011480:Protease Inhibitors; D012756:Sheep
D001580:Benzopyrenes; D011480:Protease Inhibitors; D009249:NADP; D009251:NADPH-Ferrihemoprotein Reductase; D009245:NADH Dehydrogenase