Purification and Characterization of a Membrane-Bound NADPH-Cytochrome c Reductase Capable of Catalyzing Menadione-Dependent O2- Formation in Guinea Pig Polymorphonuclear Leukocytes1

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Abstract
A membrane-bound NADPH-cytochrome c reductase, which is capable of forming the superoxide anion (O 2− ) in the presence of menadione, was highly purified from membrane fractions of disrupted guinea pig polymorphonuclear leukocytes by solubilization with 0.2% Triton X-100 and chromatographies on Sephacryl S-300 and 2′,5′-ADP-agarose. The overall purification from the membrane fraction was over 110-fold, with a yield of about 6%. The purified preparation did not contain two other pyridine nucleotide-oxidizing enzymes: NADH- and NAD(P)H-oxidizing enzymes ( J. Biochem . 94 , 931–936, 1983). Besides cytochrome c , the purified enzyme was able to reduce menadione, Nitroblue tetrazolium (NBT) and 2,6-dichlorophenolindophenol. The reduction of menadione alone resulted in the formation of O 2− . The purified enzyme preparation contained FAD. When assayed by measuring O 2− generation in the presence of menadione, the enzyme showed an optimum pH at 7.0–7.4, and K m values for NADPH, NADH, and menadione were 25, 230, and 5.3 μM, respectively. The enzyme activity was not inhibited by NaN 3 or dicumarol, but was by N -ethylmaleimide, EDTA, and quercetin; these inhibition profiles agree with those observed for the NADPH oxidase in the membrane fraction of phorbol-myristate acetate-stimulated leukocytes. Furthermore, when compared by means of the NBT-staining method combined with disc gel electrophoresis, the purified enzyme was electrophoretically indistinguishable from the NADPH-NBT reductase in the plasma membrane as well as phagosomes of the leukocytes. These results suggest that the purified NADPH-cytochrome c reductase is the putative flavoprotein of the NADPH oxidase system responsible for the respiratory burst.