Possible target components for the inhibitory effect of N-ethylmaleimide on the activation of neutrophil NADPH oxidase

Potential target components for the inhibitory effect of covalent sulfhydryl-modifying reagent N-ethylmaleimide (NEM) on the activation of NADPH oxidase in human neutrophils was studied in a cell-free system. The capacity of both cytosol and membrane fractions to induce the translocation of cytosolic components and O-2(-) generation in the cell-free activation system was affected by NEM. The phosphorylation of p47(phox), which mediates the translocation of cytosolic complex, by protein kinase C was not inhibited by NEM and NEM-treated p47(phox) was as effective as untreated p47(phox) both in the kinase-dependent and in the amphiphile-dependent cell-free activation sysyems. In addition, phosphatidic acid-dependent phosphorylation of cytosol including p47(phox) was not affected by NEM. The inhibition of cytosol's capacity to activate NADPH oxidase was partially reversed by an addition of the fraction containing G-protein vac. Taken together, the data suggest that membrane component cytochrome b(558) and cytosolic component vac may be the potential targets for the NEM effect on the activation of NADPH oxidase.