A cytosolic FAD-containing enzyme catalyzing cytochrome c reduction in Trypanosoma cruzi. I. Purification and some properties.
A cytosolic flavoprotein enzyme for the protozoan, Trypanosoma cruzi, has been purified essentially to homogeneity by DEAE-cellulose and 2',5'-ADP-agarose column chromatography. The native enzyme has a molecular weight of 100,000 +/- 6,000, is composed of two identical subunits of molecular weight 52,000 +/- 1,000, and contains FAD in the ratio of 1 mol of FAD per mol of enzyme subunit. The enzyme is NADPH-dependent and is capable of reducing cytochrome c, ferricyanide, 2,6-dichloroindophenol, and menadione, but not adrenalin. It does not hydroxylate either sodium salicylate or sodium p-hydroxybenzoate, but N-methylaniline and N,N-dimethylaminobenzaldehyde-supported oxidation of NADPH has been demonstrated. Plots of initial velocity against NADPH concentration give hyperbolic curves with Km values of 6.289 X 10(-5) M. The enzyme is clearly different from the microsomal NADPH-cytochrome c reductase in its intracellular distribution, molecular weight, dimeric nature, presence of only FAD, and activity against secondary and tertiary aromatic amines.
D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005182:Flavin-Adenine Dinucleotide / Q000302:isolation & purification*; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
D005182:Flavin-Adenine Dinucleotide; D009251:NADPH-Ferrihemoprotein Reductase