title: Conformational changes in truncated p47phox proteins monitored by fluorescent labeling.
abstract: The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the reduction of oxygen to O2- at the expense of NADPH. During activation, the cytosolic oxidase components p47phox and p67phox, each containing two Src homology 3 (SH3) domains, migrate to the plasma membrane. p47phox and p67phox associate with cytochrome b558, 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, as an activating agent. Activators of the oxidase in vitro cause exposure of the SH3 domains of p47phox, which has probably been masked by the C-terminal region of this protein in a resting state. We show here that the fluorescence exhibited by the covalently labeled N,N'-di-methyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine (IANBD) was increased when N-terminal-truncated p47Phox-(SH3)2-C was treated with anionic amphiphiles. This finding was similar to the results obtained with the full-length p47phox. However, the fluorescence of C-terminal-truncated p47Phox-N-(SH3)2 and that of both C-terminal and N-terminal truncated p47Phox-(SH3)2 were not altered by the activators. These results indicate that the C-terminal region of p47phox is a primary target of the conformational change during the activation of NADPH oxidase.
authors: Department of Biochemistry, College of Natural Sciences, Kyung-pook National University, Taegu, Korea.
authors: J H
authors: JH
authors: Lee
authors: J W
authors: JW
authors: Park
mesh_terms: D000595:Amino Acid Sequence; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000737:chemistry*; D019255:NADPH Oxidases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D010750:Phosphoproteins; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1