Kinetic and structural properties of biocatalytically active sheep lung microsomal NADPH-cytochrome c reductase.
NADPH-cytochrome c reductase was purified to electrophoretic homogeneity from detergent solubilized sheep lung microsomes. The specific activity of the purified enzyme ranged from 56 to 67 mumol cytochrome c reduced/min/mg protein and the yield was 48-52% of the initial activity in lung microsomes. The reductase had Mr of 78,000 and contained 1 mol each of FAD and FMN. Km values obtained in 0.3 M phosphate buffer, pH 7.8 at 37 degrees C for NADPH and cytochrome c were 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM. Lung reductase was inhibited by its substrate, cytochrome c when its concentration was above 160 microM. The lung reductase exhibited a ping-pong type kinetic mechanism for NADPH mediated cytochrome c reduction. Purified lung reductase was biocatalytically active in supporting benzo(a)pyrene hydroxylation reaction when coupled with lung cytochrome P-450 and lipid.
D000818:Animals; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D012756:Sheep
D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase