Enhancement by Ca2+ or Mg2+ of catalytic activity of the superoxide-producing NADPH oxidase in membrane fractions of human neutrophils and monocytes.
To examine the role of divalent cations in the generation of superoxide anion (O2-) by the NADPH oxidase system of phagocytic cells, membrane-rich fractions were prepared from human neutrophils and monocytes. O2- generation by the fractions in sucrose was enhanced by addition of Ca2+ or Mg2+. EDTA inhibited most of the O2- generation; Ca2+ or Mg2+ reversed the inhibition. Zn2+, Mn2+, or Cu2+ completely inhibited O2- production. Neutrophil membrane fraction solubilized with Triton X-100, then passed through a chelating column, lost 80% of its oxidase activity; the loss could be reversed by addition of Ca2+ or Mg2+. Addition of 0.3 mM Ca2+ or Mg2+ protected against thermal instability of the enzyme. Kinetic analysis of the neutrophil oxidase activity as a function of NADPH and Ca2+ or Mg2+ concentrations showed that cation did not interact with NADPH in solution or affect the binding of NADPH to the oxidase; rather, cation bound directly to the oxidase, or to some associated regulatory component, to activate the enzyme. For the neutrophil oxidase, the Km for NADPH was 51 +/- 6 (S.D.) microM. Hyperbolic saturation was observed with Ca2+ and Mg2+, and the Kd values were 1.9 +/- 0.3 and 2.9 +/- 0.3 microM, respectively, suggesting that the oxidase, or some associated component, has a relatively high-affinity binding site for Ca2+ and Mg2+.
Suzuki
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Pabst
M J
MJ
Johnston
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RB
D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002614:Chelating Agents; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009000:Monocytes / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D012117:Resins, Synthetic; D013481:Superoxides / Q000378:metabolism*
D002614:Chelating Agents; D012117:Resins, Synthetic; D013481:Superoxides; C024997:Chelex 100; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D008274:Magnesium; D002118:Calcium