Lee J H, Park J W
Department of Biochemistry, College of Natural Sciences, Kyung-pook National University, Taegu, Korea.
J Protein Chem. 2000 Nov;19(8):643-8. doi: 10.1023/a:1007144101361.
The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the reduction of oxygen to O2- at the expense of NADPH. During activation, the cytosolic oxidase components p47phox and p67phox, each containing two Src homology 3 (SH3) domains, migrate to the plasma membrane. p47phox and p67phox associate with cytochrome b558, a membrane-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachidonic acid, as an activating agent. Activators of the oxidase in vitro cause exposure of the SH3 domains of p47phox, which has probably been masked by the C-terminal region of this protein in a resting state. We show here that the fluorescence exhibited by the covalently labeled N,N'-di-methyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine (IANBD) was increased when N-terminal-truncated p47Phox-(SH3)2-C was treated with anionic amphiphiles. This finding was similar to the results obtained with the full-length p47phox. However, the fluorescence of C-terminal-truncated p47Phox-N-(SH3)2 and that of both C-terminal and N-terminal truncated p47Phox-(SH3)2 were not altered by the activators. These results indicate that the C-terminal region of p47phox is a primary target of the conformational change during the activation of NADPH oxidase.
中性粒细胞的白细胞NADPH氧化酶是一种膜结合酶，它以NADPH为代价催化氧气还原为超氧阴离子（O2-）。在激活过程中，胞质氧化酶成分p47phox和p67phox（各自含有两个Src同源3（SH3）结构域）迁移到质膜。p4447phox和p67phox与细胞色素b558（一种膜整合黄素血红蛋白）结合，组装成活性氧化酶。在无细胞系统中，可以使用阴离子两亲物（如十二烷基硫酸钠或花生四烯酸）作为激活剂来模拟氧化酶的激活。体外氧化酶激活剂会导致p47phox的SH3结构域暴露，在静止状态下该结构域可能被该蛋白的C末端区域所掩盖。我们在此表明，当用阴离子两亲物处理N末端截短的p47Phox-(SH3)2-C时，共价标记的N,N'-二甲基-N(碘乙酰基)-N'-(7-硝基苯并-2-恶唑-1,3-二氮杂环丁烷-4-基)乙二胺（IANBD）发出的荧光会增加。这一发现与全长p47phox得到的结果相似。然而，C末端截短的p47Phox-N-(SH)2以及C末端和N末端均截短的p47Phox-(SH3)2的荧光不会因激活剂而改变。这些结果表明，p47phox的C末端区域是NADPH氧化酶激活过程中构象变化的主要靶点。
