The O2- generating oxidoreductase of human neutrophils: evidence of an obligatory requirement for calcium and magnesium for expression of catalytic activity.
NADPH-dependent O2- generating oxidoreductase activity recovered from cell lysates of phorbol myristate acetate-stimulated human neutrophils exhibits dependence on Ca+2 and Mg+2 for full expression of its catalytic activity. O2- generating activity was completely abolished by exposure of the oxidoreductase to EDTA, then reconstituted by exposure of the enzyme to Ca+2 and Mg+2 in excess of the EDTA concentration used to block catalytic activity. The oxidoreductase responded maximally to either 0.25 mM Ca+2 or 0.80 mM Mg+2. The pH optimum of the oxidoreductase exposed to Ca+2 and Mg+2 is between pH 7.0 and 7.6. The molar ratio of NADPH oxidation to O2- production determined at pH 7.6 in the presence of Ca+2 and Mg+2 is 0.49, indicating 1 mole of NADPH oxidized per 2 moles of O2- formed. Particulate fractions recovered from cell lysates of resting neutrophils exhibited no oxidoreductase activity under the same conditions.
D002118:Calcium / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004492:Edetic Acid; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium