Fused p47phox and p67phox Truncations Efficiently Reconstitute NADPH Oxidase with Higher Activity and Stability than the Individual Components

Kentaro Ebisu, Teruaki Nagasawa, Kyoji Watanabe, Katsuko Kakinuma, Kei Miyano, Minoru Tamura

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Activation of the neutrophil NADPH oxidase occurs via assembly of the cytosolic regulatory proteins p47phox, p67phox, and Rac with the membrane-associated flavocytochrome b558. 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 Km for NADPH. O2.- 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 amphifile in a cell-free system containing cytochrome b558 relipidated with phosphatidylinositol- or phosphatidylserine-enriched phospholipid mixtures. From the results we consider multiple roles of anionic amphifiles in a cell-free activation, which could be substituted by our system. The fact that a fusion produces a more stable complex indicates that interactions among components determine the longevity of the complex. Based on the findings we propose a model for the topology among p47N, p67N, and cytochrome b558 in the active complex.

Original languageEnglish
Pages (from-to)24498-24505
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number27
DOIs
Publication statusPublished - Jul 6 2001

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NADPH Oxidase
Chemical activation
Proteins
Fusion reactions
Cell-Free System
NADP
Neutrophil Activation
Phosphatidylserines
Phosphatidylinositols
neutrophil cytosol factor 67K
Phospholipids
Topology
Membranes
cytochrome b558

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Fused p47phox and p67phox Truncations Efficiently Reconstitute NADPH Oxidase with Higher Activity and Stability than the Individual Components. / Ebisu, Kentaro; Nagasawa, Teruaki; Watanabe, Kyoji; Kakinuma, Katsuko; Miyano, Kei; Tamura, Minoru.

In: Journal of Biological Chemistry, Vol. 276, No. 27, 06.07.2001, p. 24498-24505.

Research output: Contribution to journalArticle

Ebisu, Kentaro ; Nagasawa, Teruaki ; Watanabe, Kyoji ; Kakinuma, Katsuko ; Miyano, Kei ; Tamura, Minoru. / Fused p47phox and p67phox Truncations Efficiently Reconstitute NADPH Oxidase with Higher Activity and Stability than the Individual Components. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 27. pp. 24498-24505.
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