Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex

Yukio Nisimoto, Hisamitsu Ogawa, Kei Miyano, Minoru Tamura

研究成果: ジャーナルへの寄稿記事

14 引用 (Scopus)

抄録

A series of truncated forms of His6-tagged gp91phox were expressed, solubilized, and purified in the presence of 30 μM FAD. The truncated gp91phox with the longest sequence in the C-terminal region (221-570) (gp91C) showed the highest activity (turnover rate, 0.92) for NADPH diaphorase in the presence of either 0.3% Triton X-100 or 0.5% Genapol X-80. Activity was not inhibited by superoxide dismutase but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. The flavinated gp91C contained approximately 0.9 mol of FAD/mol of protein (MW 46 kDa) and 12% α-helix content. In the absence of p47phox, p67phox showed considerable activation of gp91C in the presence of Rac. Carboxyl-terminal truncated p67phox (1-210) (p67N), which is the minimal active fragment, was fused with Rac or Q61LRac. The fusion protein p67N-Rac (or p67N-Q61LRac) showed a 2-fold higher stimulatory effect on NBT reductase activity of gp91C than the combination of the individual cytosolic p67N and Rac proteins. In contrast, Rac-p67N, a fusion with the opposite orientation, showed a smaller significant effect on the enzyme activity. The EC50 values for p67phox, p67N, p67N-Rac, and Rac-p67N were 8.00. 4.35, 2.56, and 15.2 μM, respectively, while the Km value for NADPH in the presence and absence of the cytosolic components was almost the same (40-55 μM). In the presence of Rac, p67N or p67phox bound to gp91C with a molar ratio of approximately 1:1 but neither p67N nor Rac alone showed significant binding.

元の言語英語
ページ(範囲)9567-9575
ページ数9
ジャーナルBiochemistry
43
発行部数29
DOI
出版物ステータス出版済み - 7 27 2004

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Flavoproteins
Chemical activation
Flavin-Adenine Dinucleotide
rac GTP-Binding Proteins
Fusion reactions
NADPH Dehydrogenase
Octoxynol
Enzyme activity
NADP
Superoxide Dismutase
Oxidoreductases
Proteins
neutrophil cytosol factor 67K
Enzymes

All Science Journal Classification (ASJC) codes

  • Biochemistry

これを引用

Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex. / Nisimoto, Yukio; Ogawa, Hisamitsu; Miyano, Kei; Tamura, Minoru.

:: Biochemistry, 巻 43, 番号 29, 27.07.2004, p. 9567-9575.

研究成果: ジャーナルへの寄稿記事

Nisimoto, Yukio ; Ogawa, Hisamitsu ; Miyano, Kei ; Tamura, Minoru. / Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex. :: Biochemistry. 2004 ; 巻 43, 番号 29. pp. 9567-9575.
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title = "Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex",
abstract = "A series of truncated forms of His6-tagged gp91phox were expressed, solubilized, and purified in the presence of 30 μM FAD. The truncated gp91phox with the longest sequence in the C-terminal region (221-570) (gp91C) showed the highest activity (turnover rate, 0.92) for NADPH diaphorase in the presence of either 0.3{\%} Triton X-100 or 0.5{\%} Genapol X-80. Activity was not inhibited by superoxide dismutase but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. The flavinated gp91C contained approximately 0.9 mol of FAD/mol of protein (MW 46 kDa) and 12{\%} α-helix content. In the absence of p47phox, p67phox showed considerable activation of gp91C in the presence of Rac. Carboxyl-terminal truncated p67phox (1-210) (p67N), which is the minimal active fragment, was fused with Rac or Q61LRac. The fusion protein p67N-Rac (or p67N-Q61LRac) showed a 2-fold higher stimulatory effect on NBT reductase activity of gp91C than the combination of the individual cytosolic p67N and Rac proteins. In contrast, Rac-p67N, a fusion with the opposite orientation, showed a smaller significant effect on the enzyme activity. The EC50 values for p67phox, p67N, p67N-Rac, and Rac-p67N were 8.00. 4.35, 2.56, and 15.2 μM, respectively, while the Km value for NADPH in the presence and absence of the cytosolic components was almost the same (40-55 μM). In the presence of Rac, p67N or p67phox bound to gp91C with a molar ratio of approximately 1:1 but neither p67N nor Rac alone showed significant binding.",
author = "Yukio Nisimoto and Hisamitsu Ogawa and Kei Miyano and Minoru Tamura",
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T1 - Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex

AU - Nisimoto, Yukio

AU - Ogawa, Hisamitsu

AU - Miyano, Kei

AU - Tamura, Minoru

PY - 2004/7/27

Y1 - 2004/7/27

N2 - A series of truncated forms of His6-tagged gp91phox were expressed, solubilized, and purified in the presence of 30 μM FAD. The truncated gp91phox with the longest sequence in the C-terminal region (221-570) (gp91C) showed the highest activity (turnover rate, 0.92) for NADPH diaphorase in the presence of either 0.3% Triton X-100 or 0.5% Genapol X-80. Activity was not inhibited by superoxide dismutase but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. The flavinated gp91C contained approximately 0.9 mol of FAD/mol of protein (MW 46 kDa) and 12% α-helix content. In the absence of p47phox, p67phox showed considerable activation of gp91C in the presence of Rac. Carboxyl-terminal truncated p67phox (1-210) (p67N), which is the minimal active fragment, was fused with Rac or Q61LRac. The fusion protein p67N-Rac (or p67N-Q61LRac) showed a 2-fold higher stimulatory effect on NBT reductase activity of gp91C than the combination of the individual cytosolic p67N and Rac proteins. In contrast, Rac-p67N, a fusion with the opposite orientation, showed a smaller significant effect on the enzyme activity. The EC50 values for p67phox, p67N, p67N-Rac, and Rac-p67N were 8.00. 4.35, 2.56, and 15.2 μM, respectively, while the Km value for NADPH in the presence and absence of the cytosolic components was almost the same (40-55 μM). In the presence of Rac, p67N or p67phox bound to gp91C with a molar ratio of approximately 1:1 but neither p67N nor Rac alone showed significant binding.

AB - A series of truncated forms of His6-tagged gp91phox were expressed, solubilized, and purified in the presence of 30 μM FAD. The truncated gp91phox with the longest sequence in the C-terminal region (221-570) (gp91C) showed the highest activity (turnover rate, 0.92) for NADPH diaphorase in the presence of either 0.3% Triton X-100 or 0.5% Genapol X-80. Activity was not inhibited by superoxide dismutase but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. The flavinated gp91C contained approximately 0.9 mol of FAD/mol of protein (MW 46 kDa) and 12% α-helix content. In the absence of p47phox, p67phox showed considerable activation of gp91C in the presence of Rac. Carboxyl-terminal truncated p67phox (1-210) (p67N), which is the minimal active fragment, was fused with Rac or Q61LRac. The fusion protein p67N-Rac (or p67N-Q61LRac) showed a 2-fold higher stimulatory effect on NBT reductase activity of gp91C than the combination of the individual cytosolic p67N and Rac proteins. In contrast, Rac-p67N, a fusion with the opposite orientation, showed a smaller significant effect on the enzyme activity. The EC50 values for p67phox, p67N, p67N-Rac, and Rac-p67N were 8.00. 4.35, 2.56, and 15.2 μM, respectively, while the Km value for NADPH in the presence and absence of the cytosolic components was almost the same (40-55 μM). In the presence of Rac, p67N or p67phox bound to gp91C with a molar ratio of approximately 1:1 but neither p67N nor Rac alone showed significant binding.

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