DIPHENYLENE IODONIUM AS AN INHIBITOR OF THE NADPH OXIDASE COMPLEX OF BOVINE NEUTROPHILS - FACTORS CONTROLLING THE INHIBITORY POTENCY OF DIPHENYLENE IODONIUM IN A CELL-FREE SYSTEM OF OXIDASE ACTIVATION
DIPHENYLENE IODONIUM AS AN INHIBITOR OF THE NADPH OXIDASE COMPLEX OF BOVINE NEUTROPHILS - FACTORS CONTROLLING THE INHIBITORY POTENCY OF DIPHENYLENE IODONIUM IN A CELL-FREE SYSTEM OF OXIDASE ACTIVATION
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DOI：
10.1111/j.1432-1033.1992.tb17159.x
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发表时间：
1992-08-15
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;VIGNAIS, PV
二苯碘鎓（Ph 2 I）是一种亲脂性试剂，能有效抑制牛中性粒细胞NADPH氧化酶产生O2-。在由中性粒细胞膜和来自静息细胞的胞质溶胶组成的NADPH氧化酶活化的无细胞系统中，补充有鸟苷5 '-[γ-硫代]三磷酸、MgCl 2和花生四烯酸，或在从由4-β-佛波醇12-肉豆蔻酸酯13-乙酸酯活化的中性粒细胞分离的膜中，添加还原剂，例如NADPH或连二亚硫酸钠，显著增强了Ph 2 I对NADPH氧化酶的抑制。发现膜部分含有Ph 2 I敏感组分。在Ph 2 I浓度足以完全抑制O2-生产（约10 nmol/mg膜蛋白）的存在下，添加催化量的氧化还原介体二氯靛酚（Cl 2 Ind）导致电子流旁路到细胞色素c，其速率约为非抑制氧化酶中测定的速率的一半。通过添加脱氧胆酸钠，实现了该旁路效率的显著增加。Cl 2-Ind-mediated细胞色素c的减少是可以忽略不计的膜分离从静息中性粒细胞。在较高浓度的Ph 2 I（100 nmol/mg膜蛋白），Cl 2 Ind介导的细胞色素c还原酶活性仅被抑制一半，这表明，在NADPH氧化酶复合物中，至少有两个Ph 2 I敏感的组分，不同的是它们对抑制剂的敏感性。在低浓度的Ph 2 I（< 10 nmol/mg蛋白质）下，分离的中性粒细胞膜中还原的细胞色素b558的光谱被修改，这表明对低浓度Ph 2 I敏感的组分是细胞色素b558的血红素结合组分。发现较高浓度的Ph 2 I抑制氧化酶复合物的分离的NADPH脱氢酶组分。用[I-125] Ph 2 I标记了许多膜蛋白和胞浆蛋白。然而，放射性标记的膜结合的24-kDa的蛋白质，这可能是细胞色素b558的小亚基，响应更具体的条件下的激活和还原所需的抑制O2-生产的Ph 2 I。黄嘌呤氧化酶的O2-生成形式也被Ph 2 I抑制。黄嘌呤氧化酶，一种非血红素铁黄素蛋白，抑制Ph 2 I，有一些共同的特点，与中性粒细胞NADPH氧化酶，即需要的还原条件抑制O2-生产Ph 2 I和诱导的旁路电子流细胞色素c的Cl 2 Ind在抑制酶，这表明一些相似的分子组织中的两种酶。
Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a cell-free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes isolated from neutrophils activated by 4-beta-phorbol 12-myristate 13-acetate, addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to contain the Ph2I-sensitive component(s). In the presence of a concentration of Ph2I Sufficient to fully inhibit O2- production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non-inhibited oxidase. A marked increase in the efficiency of this by-pass was achieved by addition of sodium deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph2I (100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph2I sensitive components, differing by their sensitivity to the inhibitor. At low concentrations of Ph2I (< 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph2I is the heme binding component of cytochrome b558. Higher concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [I-125]Ph2I. However, the radiolabeling of a membrane-bound 24-kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of O2- production by Ph2I. The O2--generating form of xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a non-heme iron flavoprotein, by Ph2I had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of O2- production by Ph2I and the induction of a by-pass of electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.