Inactivation of neutrophil NADPH oxidase upon dilution and its prevention by cross-link and fusion of phox proteins
| Autor: | Hiroki Kitahara, Katsuko Kakinuma, Shinobu Ohmi, Minoru Tamura, Kei Miyano |
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| Rok vydání: | 2004 |
| Předmět: |
Hot Temperature
Time Factors Cytochrome Neutrophils Blotting Western Biophysics Flavin group Biochemistry chemistry.chemical_compound Flavins Enzyme Stability Sodium dodecyl sulfate Molecular Biology Carbodiimide Oxidase test NADPH oxidase biology Superoxide NADPH Oxidases Sodium Dodecyl Sulfate Cytochromes b Fusion protein Molecular biology chemistry biology.protein |
| Zdroj: | Archives of biochemistry and biophysics. 431(1) |
| ISSN: | 0003-9861 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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