Endogenous Protein Phosphorylation by Resting and Activated Human Neutrophils By Patricia

NADPH oxidase is an enzyme in the plasma membrane of the neutrophil that catalyzes the production of 02 #{149}a species central to the oxygen-dependent killing mechanisms of this cell. The oxidase is dormant in resting cells and is activated upon the addition of a stimulus. Neutrophils of patients with chronic granulomatous disease (CGD) manifest no oxidase activity when stimulated. The possible role of protein phosphorylation in the activation of NADPH oxidase was examined in normal and CGD neutrophils by measuring the incorporation of 32P into proteins as determined by gel electrophoresis followed by autoradiography. Resting neutrophils from normal subjects exhibit at least 40 distinct phosphoprotein bands. The level of phosphorylation of these bands was examined after the addition of phorbol myristate acetate (PMA). opsonized zymosan. digitonin. N-formyl-methionyl-leucyl-phenylalanine (FMLP). or NaF. PMA and opsonized zymosan increased the phosphorylation of a set of 6 protein bands. Digitonin and FMLP consistently caused the phosphorylation of 4 of these protein bands. while NaF failed to induce increased phosphorylation of any protein band. All activators tested caused the dephosphorylation of one specific protein band.