Purification and resolution of NADH diaphorase activity from NADPH diaphorase-linked: O2 oxidoreductase activity of human neutrophils.
Intrinsic NADPH diaphorase activity is a component of the membrane-bound NAD(P)H:O2 oxidoreductase of human neutrophils. NADH-specific diaphorase activity is also present in membrane fractions rich in oxidoreductase activity. Studies were undertaken to determine whether the NADH diaphorase might also be intrinsic to the oxidoreductase. The latter diaphorase was freed from the membrane by detergent extraction and partially purified approximately 80-fold. Its apparent molecular weight following solubilization in deoxycholate and Tween-20 was 204 000 +/- 10 000. The specific activity of the partially purified diaphorase with ferricyanide as electron acceptor was 7.6 X 10(3) mU/mg protein, its pH optimum was 7.0, and its Km for NADH was 13 microM. It is completely devoid of NADPH diaphorase activity, lacks the capacity to reduce molecular oxygen, yet readily reduces ferricyanide, 2,6-dichlorophenolindophenol and ferricytochrome c. Whereas the NADH diaphorase was freed from the particulate fraction of cell lysates by extraction in 10 mM Tris-HCl buffer (pH 8.6) made up in 15% glycerol and 0.5% Tween-20, NADPH-dependent diaphorase and superoxide-generating activities also present in the membrane were not. These observations make it unlikely that the principal membrane-bound NADH diaphorase found in human neutrophils is a component of the NAD(P)H:O2 oxidoreductase, despite its common association in the same particulate fraction of cell lysates.
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003840:Deoxycholic Acid / Q000494:pharmacology; D008058:Dihydrolipoamide Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D011136:Polysorbates; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D011136:Polysorbates; D003840:Deoxycholic Acid; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme; D008058:Dihydrolipoamide Dehydrogenase; D013755:Tetradecanoylphorbol Acetate