Translocation of guinea pig p40-phox during activation of NADPH oxidase.
The superoxide-producing NADPH oxidase consists of membrane-associated cytochrome b558 and cytosolic components, p47-phox and p67-phox. Recently, we have found a novel cytosolic component, p40-phox, which is tightly associated with p67-phox. In this study, we examined the translocation of p40-phox during activation of NADPH oxidase in a cell-free system using the membrane and the purified p47-phox/p67-phox/p40-phox complex. p40-phox was translocated to the membrane by arachidonic acid in a dose-dependent manner. The translocation pattern of p40-phox was similar to those of p47-phox and p67-phox. However, immunoprecipitation assay revealed that p40-phox was dissociated from p47-phox and p67-phox during activation. The translocation of three cytosolic components was not affected by the deletion of GTP-gamma-s from the reaction mixture. Interestingly, a synthetic peptide corresponding to carboxyl-terminus of p40-phox inhibited the activation of NADPH oxidase and translocation of p40-phox, p47-phox, and p67-phox, suggesting that p40-phox might play a role in the activation of NADPH oxidase. These observations suggest that p40-phox is dissociated from p67-phox during activation, and translocates to the membrane by GTP-gamma-s-independent mechanism.
Someya
A
A
Department of Biochemistry, Juntendo University, School of Medicine, Tokyo, Japan.
Nagaoka
I
I
Nunoi
H
H
Yamashita
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006168:Guinea Pigs; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D010446:Peptide Fragments; D010750:Phosphoproteins; C085072:neutrophil cytosol factor 40K; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1