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, GTP gamma S and SDS, and found that both G-protein and GTP gamma S are essential for the activation of the NADPH oxidase. The effect of GTP gamma S is modified by Mg2+: the cations enhance the O2- generation at low concentrations of GTP gamma S, whereas they attenuate the activity at higher concentrations of GTP gamma S. In presence of 10 microM GTP gamma S, the maximal activity is observed at 0.1 microM 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.
Aoyagi
K
K
Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan.
Takeshige
K
K
Sumimoto
H
H
Nunoi
H
H
Minakami
S
S
D000911:Antibodies, Monoclonal; D003600:Cytosol / Q000201:enzymology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D015636:Magnesium Chloride / Q000494:pharmacology*; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D013481:Superoxides / Q000097:blood*; D013873:Thionucleotides / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D013873:Thionucleotides; D015636:Magnesium Chloride; D013481:Superoxides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D004492:Edetic Acid; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D019204:GTP-Binding Proteins