Cell-Free Activation of the Respiratory Burst Oxidase by Protein Kinase C

In intact neutrophils, phorbol ester treatment activates the respiratory burst oxidase, the enzyme responsible for O2-production by phagocytes. This effect is thought to be dependent on protein kinase C and on the phosphorylation of p47phox. In this paper, we report that protein kinase C activates the respiratory burst oxidase in a cell-free system consisting of isolated neutrophil cytosol and membrane. Oxidase activation required a highly active protein kinase C, recombinant p47phox and ATP, and was inhibited by the protein kinase C inhibitors H-7 and GF-109203X. Partial depletion of cytosolic ATP by dialysis reduced oxidase activation by over 50%. In contrast, neither protein kinase C inhibitors nor ATP depletion affected oxidase activation by SDS. These findings strongly suggest that in the cell-free system, the oxidase can be activated by the phosphorylation of p47phox.