Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex
Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex
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DOI：
10.1021/bi0400249
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发表时间：
2004-07-27
期刊：
影响因子：
2.9
通讯作者：
Tamura, M
中科院分区：
文献类型：
--
作者：
Nisimoto, Y;Ogawa, H;Tamura, M
在30um的FAD存在下，表达、溶解和纯化了一系列截短形式的His(6)标记的gp91Phox。在0.3%Triton X-100或0.5%Genapol X-80存在下，C末端(221-570)最长序列(Gp91C)的截短gp91Phox对NADPH黄递酶的活性最高(转化率为0.92)。超氧化物歧化酶不抑制该酶的活性，但呼吸爆发氧化酶的抑制剂二苯基碘可阻断该酶的活性。调味的gp91C含有约0.9摩尔的FAD/摩尔蛋白质(相对分子质量为46 kDa)和12%的α-螺旋含量。在没有p47Phox的情况下，p67Phox在RAC存在的情况下表现出相当大的gp91C活性。将最小活性片段--羧基末端截短的p67Phox(1-210)(P67N)与Rac或Q61LRac融合。融合蛋白p67N-Rac(或p67N-Q61LRac)对gp91C的NBT还原酶活性的刺激作用比胞内单独的p67N和Rac蛋白高2倍。相反，定向相反的融合基因RAC-p67N对酶活性的影响较小。P67Phox、p67N、p67N-RAC和RAC-p67N的EC50值为8.00。分别为4.35、2.56和15.2微米，而NADPH在胞液成分存在和不存在时的K-m值几乎相同(40-55微米)。在RAC存在下，p67N或p67Phox与gp91C以约1：1的摩尔比结合，但单独使用p67N和RAC均不显示明显的结合作用。
A series of truncated forms of His(6)-tagged gp91phox were expressed, solubilized, and purified in the presence of 30 muM 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 EC50 values for p67phox, p67N, p67N-Rac, and Rac-p67N were 8.00. 4.35, 2.56, and 15.2 muM, respectively, while the K-m value for NADPH in the presence and absence of the cytosolic components was almost the same (40-55 muM). 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.