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

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20 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)9567-9575
Number of pages9
Issue number29
Publication statusPublished - Jul 27 2004
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry


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