C-terminal region of the cytosolic subunit p47(phox) is a primary target of conformational change during the activation of leukocyte NADPH oxidase.
The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O(2(-)) from oxygen using NADPH as the electron donor. During activation, the cytosolic oxidase components p47(phox) and p67(phox), each containing two Src homology 3 (SH3) domains, migrate to the plasma membrane, where they associate with cytochrome b(558), a membrane-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachidonic acid and the phosphorylation of p47(phox )with protein kinase C. Activators of the oxidase in vitro cause exposure of p47(phox)-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. We show here that the total protein steady-state intrinsic fluorescence exhibited by the tryptophan residues of p47(phox) substantially decreased when N-terminal truncated p47(phox)-SH3-C was treated with anionic amphiphiles or phosphorylated with protein kinase C. This finding was similar to the results obtained with full-length p47(phox). However, the fluorescence of C-terminal truncated p47(phox)-N-SH3 and both C-terminal and N-terminal truncated p47(phox)-SH3 were not altered by the activators. These results indicate that the C-terminal region of p47(phox) is a primary target of the conformational change during the activation of NADPH oxidase.
Lee
J H
JH
Department of Biochemistry, College of Natural Sciences, Kyungpook National University, 702-701, Taegu, South Korea.
Lee
K S
KS
Chung
T
T
Park
J
J
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D019255:NADPH Oxidases / Q000097:blood*; D009504:Neutrophils / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000097:blood* / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D021122:Protein Subunits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
D010446:Peptide Fragments; D010750:Phosphoproteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D011493:Protein Kinase C