Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex.
A series of truncated forms of His(6)-tagged gp91phox were expressed, solubilized, and purified in the presence of 30 microM 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% alpha-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 EC(50) values for p67phox, p67N, p67N-Rac, and Rac-p67N were 8.00. 4.35, 2.56, and 15.2 microM, respectively, while the K(m) value for NADPH in the presence and absence of the cytosolic components was almost the same (40-55 microM). 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.
Nisimoto
Yukio
Y
Department of Biochemistry, Aichi Medical University, School of Medicine, Nagakute, Aichi 480-1195, Japan. nisiio@aichi-med-u.ac.jp
Ogawa
Hisamitsu
H
Miyano
Kei
K
Tamura
Minoru
M
D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
D005420:Flavoproteins; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D020744:rac GTP-Binding Proteins