Destabilization of neutrophil NADPH oxidase by ATP and other trinucleotides and its prevention by Mg(2+).
Neutrophil NADPH oxidase (O(2)(-) generating enzyme) activated in a cell-free system was deactivated by dilution. When ATP was included in dilution the deactivation was further accelerated. The deactivation by dilution was biphasic, and the half-life of the enzyme was significantly shortened by ATP in each phase. ADP and AMP had little effect on the enzyme longevity while GTP and CTP had a similar effect to ATP. Staurosporine, a wide-range inhibitor of protein kinases, had no effect on ATP-induced deactivation, suggesting that the effect was not due to a protein phosphorylation. Mg(2+) addition largely prevented the deactivation by ATP. Chemical crosslinking of the activated oxidase prevented the deactivation by dilution and ATP, suggesting that the deactivation is caused by dissociation of the oxidase complex. Estimation of actin filament (F-actin) showed that the F-actin level was markedly reduced by addition of ATP. The ATP effect on the deactivation was not prominent in a semi-recombinant system which does not contain cytosol. These results suggest that ATP-induced deactivation is largely due to the chelation of Mg(2+) and are consistent with the concept that Mg(2+) stabilizes the oxidase complex by stabilizing F-actin.
Tamura
M
M
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan. miketamu@en3.ehime-u.ac.jp
Kanno
M
M
Kai
T
T
D000199:Actins / Q000032:analysis; D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002413:Cations, Divalent; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006207:Half-Life; D006801:Humans; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*
D000199:Actins; D002413:Cations, Divalent; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D019255:NADPH Oxidases; D008274:Magnesium