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 O2-generation in the cell-free activation system was affected by NEM. The phosphorylation of p47phox, which mediates the translocation of cytosolic complex, by protein kinase C was not inhibited by NEM and NEM-treated p47phox was as effective as untreated p47phox both in the kinase-dependent and in the amphiphile-dependent cell-free activation systems. In addition, phosphatidic acid-dependent phosphorylation of cytosol including p47phox 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 rac. Taken together, the data suggest that membrane component cytochrome b558 and cytosolic component rac may be the potential targets for the NEM effect on the activation of NADPH oxidase.
Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu, Korea. parkjw@kyungpook.ac.kr
J W
JW
Park
H S
HS
Park
S M
SM
Lee
D002474:Cell-Free System; D003573:Cytochrome b Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D010712:Phosphatidic Acids / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D020744:rac GTP-Binding Proteins
D003573:Cytochrome b Group; D010712:Phosphatidic Acids; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; C036888:cytochrome b558; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D011494:Protein Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins; D005033:Ethylmaleimide