Fused p47phox and p67phox truncations efficiently reconstitute NADPH oxidase with higher activity and stability than the individual components

The Journal of Biological Chemistry
Kentaro EbisuM Tamura

Abstract

Activation of the neutrophil NADPH oxidase occurs via assembly of the cytosolic regulatory proteins p47(phox), p67(phox), and Rac with the membrane-associated flavocytochrome b(558). Following cell-free activation, enzymatic activity is highly labile (Tamura, M., Takeshita, M., Curnutte, J. T., Uhlinger, D. J., and Lambeth, J. D. (1992) J. Biol. Chem. 267, 7529-7538). To try to stabilize the activity and investigate the nature of the complex, fusion proteins between p47N-(1-286) and p67N-(1-210) were constructed. In a cell-free system, a fusion protein, p67N-p47N, had an 8-fold higher efficiency and produced a higher activity than the individual proteins, and also resulted in an 8-fold improved efficiency for Rac and a lowered K(m) for NADPH. O(2) generating activity was remarkably stabilized by using p67N-p47N. The cytosolic proteins fused in the opposite orientation, p47N-p67N, showed similar activity and stability as individual proteins, but with a 4-fold improved efficiency compared with the individual cytosolic factors. In the system efficiency for Rac and affinity for NADPH were also higher than those with the nonfused components. Interestingly, the p67N-p47N showed nearly full activation in the absence of an anionic amph...Continue Reading

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Citations

Aug 5, 2004·Histochemistry and Cell Biology·William M Nauseef
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Nov 16, 2004·Biochemical and Biophysical Research Communications·Farid Bouzidi, Jacques Doussiere
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Sep 29, 2011·Journal of Leukocyte Biology·Asma TliliOliver Nüsse
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