Production of superoxide and hydrogen peroxide by an NADH-oxidase in guinea pig polymorphonuclear leukocytes. Modulation by nucleotides and divalent cations.

An NADH-oxidase extracted from granulocytes with isotonic KC1 catalyzes a cyanide insensitive production of HzOz. The stoichiometry of the reaction is: NADH + H+ + O2 + NAD+ + HzOZ. The K, (NADH) of this reaction is 0.4 mM and the maximum velocity (9.72 + 1.66 S.D. nmol of Oz/min/107 cells) is sufficient to account for the net oxygen uptake observed during phagocytosis of a wide variety of particles. The rates of peroxide production by 15 other potential electron donors are ~15% of the rate with NADH. The apparent molecular weight is approximately 310,000 + 14,000. Superoxide is produced during the reaction and dismutates to peroxide. The stoichiometry of NADH oxidation and superoxide production is indicative of two pathways of electron release from the enzyme: a univalent pathway resulting in superoxide generation, and a divalent pathway which leads directly to peroxide formation. Under assay conditions, approximately 15% of the total electron flux results in superoxide formation. The K,,, for the formation of superoxide is identical to that observed for the oxidation of NADH. The oxidase is potently inhibited by a variety of nucleotides (e.g. ATP, ADP, AMP) and other anions (e.g. PPi, citrate). In the case of ATP, the inhibition is competitive with respect to the substrate NADH with a Ki of 20 pM. Divalent cations can reverse the inhibition induced by either ATP or ADP in a fashion that suggests the free (unchelated) forms of the nucleotides are the major inhibitory species. These data are discussed in terms of the known biochemical events that accompany the increased respiration observed during phagocytosis.