Purification and characterization of NADPH–cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania)

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Abstract
NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose-pyrophosphate-washed microsomal fractions from southern armyworm (S. eridania) larval midguts. The enzyme has a MW of 70,035 .+-. 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 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. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 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. These are discussed in relation to possible differences in the active sites of the enzymes.