Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).
1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005415:Flavins / Q000032:analysis; D007422:Intestines / Q000201:enzymology*; D007700:Kinetics; D007814:Larva; D007915:Lepidoptera / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D051381:Rats; D012995:Solubility
D005415:Flavins; D000249:Adenosine Monophosphate; D009251:NADPH-Ferrihemoprotein Reductase