A reassessment of the product specificity of the NADPH:O2 oxidoreductase of human neutrophils.
Native ferricytochrome c, but not acetylated ferricytochrome c, stimulates the flow of electron equivalents passing through the neutrophil NADPH:O2 oxidoreductase complex. At 28 mM it increases NADPH oxidase activity by 157 +/- 15% (n = 5) over that measured in its absence. Enhanced activity is predominantly seen in oxidoreductase-rich 27,000 X g membrane preparations obtained from phorbol myristate acetate activated cells. Superoxide formation is also enhanced. Although some of the stimulatory activity seen with addition of native ferricytochrome c to oxidoreductase-rich membrane suspensions might have been explained in terms of mitochondrial contamination, this was ruled out. Comparable membrane preparations from resting cells were devoid of NADPH oxidase activity. Azide, a well-known inhibitor of the electron transport chain, did not block the enhancing effect of native ferricytochrome c. These results indicate that native ferricytochrome c is not a suitable scavenger of superoxide in quantitating the product specificity of the oxidoreductase since it amplifies the apparent rate of superoxide formation with respect to measured rates of NADPH oxidation conducted in its absence. By using acetylated ferricytochrome c in place of native ferricytochrome c in quantitating the product specificity of the oxidoreductase we show that no more than 70% of the electron equivalents donated by NADPH to the oxidoreductase are involved in superoxide formation. The remaining 30% of the electron equivalents given up by NADPH to the oxidoreductase appear to be involved in direct formation of hydrogen peroxide.
D000107:Acetylation; D003574:Cytochrome c Group / Q000378:metabolism; D004579:Electron Transport; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008566:Membranes / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013481:Superoxides / Q000096:biosynthesis*
D003574:Cytochrome c Group; D013481:Superoxides; D009249:NADP; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases