花生四烯酸对中性粒细胞NADPH氧化酶激活的动力学研究。花生四烯酸与苯胂氧化物的拮抗作用。

Kinetic study of the activation of the neutrophil NADPH oxidase by arachidonic acid. Antagonistic effects of arachidonic acid and phenylarsine oxide.

作者信息

Doussiere J, Bouzidi F, Poinas A, Gaillard J, Vignais P V

机构信息

Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR 314 CEA-CNRS), Département de Biologie Moléculaire et Structurale, Grenoble, France.

出版信息

Biochemistry. 1999 Dec 7;38(49):16394-406. doi: 10.1021/bi991502w.

DOI:10.1021/bi991502w

PMID:10587465

Abstract

The O(2)(-) generating NADPH oxidase complex of neutrophils comprises two sets of components, namely a membrane-bound heterodimeric flavocytochrome b which contains the redox centers of the oxidase and water-soluble proteins of cytosolic origin which act as activating factors of the flavocytochrome. The NADPH oxidase can be activated in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils in the presence of GTPgammaS and arachidonic acid. NADPH oxidase activation is inhibited by phenylarsine oxide (PAO), a sulfhydryl reagent for vicinal or proximal thiol groups. The site of action of PAO was localized by photolabeling in the beta-subunit of flavocytochrome b [Doussière, J., Poinas, A, Blais, C., and Vignais, P. V. (1998) Eur. J. Biochem. 251, 649-658]. Moreover, the spin state of heme b is controlled by interaction of arachidonic acid with the flavocytochrome b [Doussière, J., Gaillard, J., and Vignais, P. V. (1996) Biochemistry 35, 13400-13410]. Here we report that the promoting effect of arachidonic acid on the activation of NADPH oxidase is due to specific binding of arachidonic acid to flavocytochrome b. Elicitation of NADPH oxidase activity by arachidonic acid is in part associated with an increased affinity of flavocytochrome b for O(2), an effect that was counteracted by the methyl ester of arachidonic acid. On the other hand, the affinity for NADPH was not affected by arachidonic acid. We further demonstrate that PAO antagonizes the effect of arachidonic acid on oxidase activation by decreasing the affinity of the oxidase for O(2), but not for NADPH. PAO induced a change in the spin state of heme b, as arachidonic acid does, with, however, some differences in the constraints imposed to the heme. It is concluded that the opposite effects of arachidonic acid and PAO are exerted on the beta-subunit of flavocytochrome b at two different interacting sites.

摘要

中性粒细胞产生超氧阴离子（O₂⁻）的NADPH氧化酶复合物由两组成分组成，即一种膜结合的异二聚体黄素细胞色素b，它含有氧化酶的氧化还原中心，以及来源于胞质溶胶的水溶性蛋白质，这些蛋白质作为黄素细胞色素的激活因子。在存在GTPγS和花生四烯酸的情况下，NADPH氧化酶可以在由静息中性粒细胞的质膜和胞质溶胶组成的无细胞体系中被激活。NADPH氧化酶的激活受到苯胂氧化物（PAO）的抑制，PAO是一种针对邻位或近端巯基的巯基试剂。通过光标记将PAO的作用位点定位在黄素细胞色素b的β亚基上[杜西埃，J.，波伊纳斯，A，布莱斯，C.，和维尼亚斯，P. V.（1998年）《欧洲生物化学杂志》251，649 - 658]。此外，血红素b的自旋状态受花生四烯酸与黄素细胞色素b相互作用的控制[杜西埃，J.，加亚尔，J.，和维尼亚斯，P. V.（1996年）《生物化学》35，13400 - 13410]。在此我们报告，花生四烯酸对NADPH氧化酶激活的促进作用是由于花生四烯酸与黄素细胞色素b的特异性结合。花生四烯酸引发NADPH氧化酶活性部分与黄素细胞色素b对O₂亲和力的增加有关，花生四烯酸甲酯可抵消这种作用。另一方面，对NADPH的亲和力不受花生四烯酸影响。我们进一步证明，PAO通过降低氧化酶对O₂而非NADPH的亲和力来拮抗花生四烯酸对氧化酶激活的作用。PAO像花生四烯酸一样诱导血红素b自旋状态的改变，然而，对血红素施加的限制存在一些差异。得出的结论是，花生四烯酸和PAO的相反作用是在黄素细胞色素b的β亚基上的两个不同相互作用位点发挥的。