Studies on the nature and activation of O2- -forming NADPH oxidase of leukocytes. II. Relationships between phosphorylation of a component of the enzyme and oxidase activity.
The activation of O2- -formation by neutrophil NADPH oxidase is associated with phosphorylation of several membrane and cytosolic proteins. In the membranes a phosphoprotein of 32 kDa belonging to the NADPH oxidase-cytochrome b-245 system (P. Bellavite et al., Free Rad. Res. Commun., 1, 11 (1985] showed the highest relative increase of 32Pi incorporation. Concomitant with the phosphorylation, a shift of the apparent molecular mass of the protein from 31 to 32 kDa occurred. The time-course, the sensitivity to trifluoperazine and the dose-dependence of phosphorylation were similar to those of O2- forming activity, except that the latter showed a longer lag-time than the former. The increase of the 32 kDa phosphoprotein was also comparable to the kinetics of cytochrome b-245 reduction by anaerobically activated neutrophils. The phosphorylation and the NADPH oxidase were triggered by various stimulants including phorbol myristate acetate, opsonized zymosan, arachidonic acid and sodium fluoride. With arachidonic acid the O2- formation was highly active but the phosphorylation was low. With fluoride the enzyme activity was reversible upon removal of the stimulant but the phosphorylation of the 32 kDa peptide was not reversible. Neutrophils treated with PMA at 17 degrees C showed phosphorylation but not activation. The results indicate that phosphorylation of a component of NADPH oxidase is a fundamental but probably not sufficient event in the activation mechanism of the enzyme.
Bellavite
P
P
Institute of General Pathology, University of Verona, Italy.
Dusi
S
S
Cassatella
M A
MA
D000693:Anaerobiosis; D000818:Animals; D003573:Cytochrome b Group / Q000097:blood; D003576:Electron Transport Complex IV / Q000378:metabolism; D004789:Enzyme Activation; D005609:Free Radicals; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D009504:Neutrophils / Q000201:enzymology*; D010085:Oxidative Phosphorylation; D013552:Swine
D003573:Cytochrome b Group; D005609:Free Radicals; D009247:NADH, NADPH Oxidoreductases; D003576:Electron Transport Complex IV