Role of Mg²⁺ in activation of nadph oxidase of human neutrophils: Evidence that Mg²⁺ acts through G-protein

The membrane fraction and three cytosolic proteins of neutrophils, p47-phox, p67-phox and a G-protein, are involved in the cell-free activation of the O₂^--generating NADPH oxidase in the presence of SDS, though it has been controversial whether the G-protein is required or just enhancing the activity. We have used the three cytosolic factors, the solubilized membrane fraction, GTPγS and SDS, and found that both G-protein and GTPγS are essential for the activation of the NADPH oxidase. The effect of GTPγS is modified by Mg²⁺: the cations enhance the O₂^- generation at low concentrations of GTPγS, whereas they attenuate the activity at higher concentrations of GTPγS. In the presence of 10 μM GTPγS, the maximal activity is observed at 0.1 μM Mg²⁺, which is several-fold higher than that at 1 mM Mg²⁺. The omission of Mg²⁺ followed by the chelation with EDTA results in loss of the activation, which is completely restored by the addition of Mg²⁺. Thus, Mg²⁺ seems to modulate the activation of the NADPH oxidase at the level of the G-protein.