Stoichiometry of O2 metabolism and NADPH oxidation of the cell-free latent oxidase reconstituted from cytosol and solubilized membrane from resting human neutrophils.

Abstract

The stoichiometry of NADPH-dependent O2 consumption expressed by reconstituted latent oxidase obtained by combining cytosol and membrane fractions from resting human neutrophils with GTP gamma S and SDS in a cell-free assay was evaluated with regard to NADPH consumed and superoxide and H2O2 production. Oxidase activity monitored simultaneously by O2 uptake analysis using a Clark cell and O2 electrode for O2 consumption, and spectrally at 340 nm for NADPH oxidation, in the presence of excess superoxide dismutase and catalase, yielded an O2 uptake: NADPH consumption ratio of 0.51 +/- 0.04 (+/- 1 S.D., n = 6). In the presence of varying concentrations of ferricytochrome c in excess of 100 microM, and with exclusion of superoxide dismutase, the net rate of O2 consumption plateaued at approximately 6% of the rate seen with exclusion of ferricytochrome c from final assay mixtures. Cytosol and solubilized membrane fractions employed in these assays were devoid of endogenous superoxide trapping, or dismutase-like, activities. These results indicate that of the total O2 consumed, 94% is associated with direct univalent generation of superoxide. The remaining albeit low level of O2 consumption appears to be recovered in the form of H2O2 indicating that the cell-free oxidase reconstituted with SDS is capable of channeling electron equivalents through its redox sites in a highly controlled manner in ensuring that superoxide is its principal O2 reduction product concomitant with oxidation of NADPH.