The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils
The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils
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DOI：
10.1046/j.1432-1033.2002.03002.x
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发表时间：
2002-07-01
期刊：
影响因子：
--
通讯作者：
Vignais, PV
中科院分区：
文献类型：
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作者：
Doussiere, J;Bouzidi, F;Vignais, PV
在之前的一项研究中，S100A8/A9 蛋白是一种 Ca2+ - 和花生四烯酸结合蛋白，在中性粒细胞胞质中含量丰富，被发现可通过胞质 phox 蛋白 p67phox 增强中性粒细胞中 O-2 (-) 生成氧化酶的氧化还原成分（即膜结合黄细胞色素 b ）的激活， p47phox 和 Rac (Doussiere J., Bouzidi F. 和 Vignais P.V. (2001) Biochem. Biophys. Res. Commun. 285, 1317-1320)。这促使我们通过免疫沉淀和蛋白质分级来检查胞质 phox 蛋白是否可以与 S100A8/A9 结合。将未活化的牛中性粒细胞的胞质提取物与结合有抗 p67phox 抗体的蛋白 A-Sepharose 一起温育后，回收的免疫沉淀物含有 S100 蛋白、p47phox 和 p67phox。胞浆蛋白分级分离包括对羟基磷灰石和 DEAE 纤维素进行两个连续的色谱步骤，然后进行等电聚焦。 S100A8/A9异二聚体蛋白与胞质phox蛋白共迁移，更具体地与p67phox和Rac2共迁移，而分离的S100A8蛋白表现出与p47phox结合的倾向。使用由重组 p67phox、p47phox 和 Rac2、中性粒细胞膜和花生四烯酸组成的半重组无细胞氧化酶激活系统，我们发现引起的氧化酶活性的 S100A8/A9 依赖性增加与膜结合黄细胞色素 b 周转的增加相对应，但与 NADPH 亲和力的变化无关 或 O-2 。在不存在 S100A8/A9 的情况下，当胞质 phox 蛋白的临界阈值浓度以上时，氧化酶激活就会偏离迈克尔动力学。将 S100A8/A9 添加到无细胞系统中使动力学完全迈克尔式。 S100A8/A9 结合胞质 phox 蛋白的倾向，以及 S100A8/A9 对氧化酶激活动力学的影响，表明 S100A8/A9 可能是胞质 phox 蛋白的支架蛋白，或者可能有助于将花生四烯酸递送至氧化酶，从而有利于胞质 phox 蛋白与氧化酶的有效相互作用。 膜结合黄细胞色素 b .
In a previous study, the S100A8/A9 protein, a Ca2+ - and arachidonic acid-binding protein, abundant in neutrophil cytosol, was found to potentiate the activation of the redox component of the O-2 (-) generating oxidase in neutrophils, namely the membrane-bound flavocytochrome b , by the cytosolic phox proteins p67phox, p47phox and Rac (Doussiere J., Bouzidi F. and Vignais P.V. (2001) Biochem. Biophys. Res. Commun. 285 , 1317-1320). This led us to check by immunoprecipitation and protein fractionation whether the cytosolic phox proteins could bind to S100A8/A9. Following incubation of a cytosolic extract from nonactivated bovine neutrophil with protein A-Sepharose bound to anti-p67phox antibodies, the recovered immunoprecipitate contained the S100 protein, p47phox and p67phox. Cytosolic protein fractionation comprised two successive chromatographic steps on hydroxyapatite and DEAE cellulose, followed by isoelectric focusing. The S100A8/A9 heterodimeric protein comigrated with the cytosolic phox proteins, and more particularly with p67phox and Rac2, whereas the isolated S100A8 protein displayed a tendancy to bind to p47phox. Using a semirecombinant cell-free system of oxidase activation consisting of recombinant p67phox, p47phox and Rac2, neutrophil membranes and arachidonic acid, we found that the S100A8/A9-dependent increase in the elicited oxidase activity corresponded to an increase in the turnover of the membrane-bound flavocytochrome b , but not to a change of affinity for NADPH or O-2 . In the absence of S100A8/A9, oxidase activation departed from michaelian kinetics above a critical threshold concentration of cytosolic phox proteins. Addition of S100A8/A9 to the cell-free system rendered the kinetics fully michaelian. The propensity of S100A8/A9 to bind the cytosolic phox proteins, and the effects of S100A8/A9 on the kinetics of oxidase activation, suggest that S100A8/A9 might be a scaffold protein for the cytosolic phox proteins or might help to deliver arachidonic acid to the oxidase, thus favoring the productive interaction of the cytosolic phox proteins with the membrane-bound flavocytochrome b .