Vergnaud S, Paclet M H, El Benna J, Pocidalo M A, Morel F
GREPI, Laboratoire d'Enzymologie, CHU, Grenoble, France; INSERM U. 479, CHU, Bichat, Paris, France.
Eur J Biochem. 2000 Feb;267(4):1059-67. doi: 10.1046/j.1432-1327.2000.01097.x.
Chronic granulomatous disease (CGD) is due to a functional defect of the O2- generating NADPH oxidase of phagocytes. Epstein-Barr-virus-immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein-Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic-activating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patients. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67-phox-deficient patients with recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40-phox from p67-phox for efficient oxidase activity. In the process, p40-phox could function as a negative regulatory factor and stabilize the resting state.
慢性肉芽肿病（CGD）是由于吞噬细胞中产生O₂的NADPH氧化酶功能缺陷所致。爱泼斯坦-巴尔病毒永生化的B淋巴细胞表达氧化酶的所有成分，其活性比中性粒细胞低100倍。与中性粒细胞一样，爱泼斯坦-巴尔病毒永生化的B淋巴细胞的氧化酶活性在不同形式的CGD中均表现为缺陷；这些细胞被用作两名p67-吞噬细胞氧化还原蛋白缺陷型CGD患者互补研究的模型。通过使用一种异源无细胞测定法在体外重建氧化酶活性，该测定法由与来自健康或CGD患者的胞质溶胶或分离的胞质激活因子（p67-吞噬细胞氧化还原蛋白、p47-吞噬细胞氧化还原蛋白、p40-吞噬细胞氧化还原蛋白）相关的膜悬浮或溶解并纯化的细胞色素b558组成。在p67-吞噬细胞氧化还原蛋白缺陷型CGD患者中，两种胞质因子缺乏或缺失：p67-吞噬细胞氧化还原蛋白和p40-吞噬细胞氧化还原蛋白。用重组p67-吞噬细胞氧化还原蛋白补充p67-吞噬细胞氧化还原蛋白缺陷患者的胞质溶胶，氧化酶活性的恢复不超过20%。相反，当在无细胞测定中，将胞质因子与从中性粒细胞膜纯化的细胞色素b558分离后添加，而不是添加胞质溶胶时，观察到氧化酶活性完全恢复。此外，同时添加重组p67-吞噬细胞氧化还原蛋白和重组p40-吞噬细胞氧化还原蛋白以p40-吞噬细胞氧化还原蛋白剂量依赖性过程逆转了先前的互补作用。这些结果表明，在氧化酶活性的重建中，p67-吞噬细胞氧化还原蛋白是限制因素；互补效率取决于膜组织和胞质环境。在体外，氧化酶从静止状态转变为激活状态是由组装导致的，这需要p40-吞噬细胞氧化还原蛋白与p67-吞噬细胞氧化还原蛋白解离以实现有效的氧化酶活性。在这个过程中，p40-吞噬细胞氧化还原蛋白可能作为负调节因子并稳定静止状态。
