Inactivation of neutrophil NADPH oxidase upon dilution and its prevention by cross-link and fusion of phox proteins.
Activation of the phagocyte NADPH oxidase involves assembly of p47(phox), p67(phox), Rac, and flavocytochrome b(558), and the activation can be triggered in a cell-free system with an anionic amphiphile. We find that the activated oxidase in a pure cell-free system was rapidly inactivated upon dilution. When the activated oxidase was treated with a chemical cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the half-life of the oxidase in dilution was extended from 1min to 4h at 25 degrees C. The cross-linked oxidase was resistant to inhibition by inactive flavin analogs, indicating that cross-linking prevents flavin exchange. When a fusion protein p67N-p47N plus RacQ61L was added, flavocytochrome b(558) became spontaneously active. Cross-linking of this mixture produced an oxidase that was extremely stable to dilution (t(1/2)=6.6h). Western blotting analysis showed the presence of a cross-link between p67N-p47N and RacQ61L. These results suggest that covalently linked phox components prevents FAD loss and stabilizes the longevity of the stoichiometric complex, extending the lifespan of the active oxidase.
Miyano
Kei
K
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan.
Kitahara
Hiroki
H
Ohmi
Shinobu
S
Kakinuma
Katsuko
K
Tamura
Minoru
M
D015153:Blotting, Western; D045303:Cytochromes b / Q000378:metabolism; D004795:Enzyme Stability; D005415:Flavins / Q000378:metabolism; D006358:Hot Temperature; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D013997:Time Factors
D005415:Flavins; D012967:Sodium Dodecyl Sulfate; D045303:Cytochromes b; D019255:NADPH Oxidases