Tsunawaki S, Kagara S, Yoshikawa K, Yoshida L S, Kuratsuji T, Namiki H
National Children's Medical Research Center, Tokyo, Japan.
J Exp Med. 1996 Sep 1;184(3):893-902. doi: 10.1084/jem.184.3.893.
Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91phox and p22phox subunits) and two cytosolic factors (p47phox and p67phox), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40phox, as a molecule that associates with p67phox in human neutrophils. Although it has been found that this p40phox protein is defective in patients with chronic granulomatous disease (CGD) who lack p67phox, evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40phox as well as against the COOH-terminal polypeptide of p67phox to examine the mode of interaction between p40phox and p67phox in a complex. The antibody against the COOH terminus of p67phox was able to communoprecipitate p40phox in conjunction with p67phox itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40phox completely dissociated the p67phox molecule from the p40phox-p67phox complex unit without any detectable coimmunoprecipitation of p67phox, despite their tight association, whereas that against the NH2 terminus of p40phox had absolutely no dissociation effect. Similar results were found regarding their effects on the O2-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40phox. However, this dissociation did not affect the translocation of the cytosolic components including p47phox to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40phox-p67phox complex. These results provide the first demonstration that p40phox is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67phox.
吞噬细胞NADPH氧化酶在静息细胞中处于休眠状态，细胞受刺激时被激活，产生超氧阴离子，这是杀菌性氧化剂的前体。活性NADPH氧化酶以酶复合物的形式存在于细胞膜上，由膜整合细胞色素b558（gp91phox和p22phox亚基）和两个胞质因子（p47phox和p67phox）组成，后两者各自含有两个src同源3（SH3）结构域。最近，我们通过放射性方法鉴定出第三个胞质因子p40phox，它是一种在人类中性粒细胞中与p67phox结合的分子。尽管已经发现缺乏p67phox的慢性肉芽肿病（CGD）患者的这种p40phox蛋白存在缺陷，但迄今为止，尚无证据表明它与NADPH氧化酶系统存在功能关联。在本研究中，我们分别制备了针对p40phox的COOH末端和NH2末端多肽以及p67phox的COOH末端多肽的抗体，以研究复合物中p40phox与p67phox的相互作用方式。正如预期的那样，针对p67phox的COOH末端的抗体能够与p67phox自身一起共沉淀p40phox。然而，非常有趣的是，针对p40phox的COOH末端的抗体完全将p67phox分子从p40phox - p67phox复合单元中解离出来，没有检测到p67phox的共免疫沉淀，尽管它们紧密结合，而针对p40phox的NH2末端的抗体则完全没有解离作用。在无细胞激活系统中，关于它们对胞质溶胶产生O2能力的影响也发现了类似结果，即COOH末端抗体有抑制作用，而针对p40phox的NH2末端的抗体则没有。然而，这种解离并不影响包括p47phox在内的胞质成分向膜的转运。一旦NADPH氧化酶被激活，针对COOH末端的抗体对激活酶的催化作用没有任何抑制作用。NADPH氧化酶的刺激剂MA和SDS不会使p40phox - p67phox复合物解离。这些结果首次证明，p40phox实际上通过其COOH末端而非NH2末端与p67phox的结合参与NADPH氧化酶的激活。
