Selective detection of NADPH oxidase in polymorphonuclear cells by means of NAD(P)H-based fluorescence lifetime imaging.
Selective detection of NADPH oxidase in polymorphonuclear cells by means of NAD(P)H-based fluorescence lifetime imaging.
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DOI：
10.1155/2008/602639
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Gunzer M
中科院分区：
文献类型：
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作者：
Niesner R;Narang P;Spiecker H;Andresen V;Gericke KH;Gunzer M
NADPH 氧化酶 (NOX2) 是一种多亚基膜结合酶复合物，在活化细胞中组装后，催化游离氧还原为其超氧阴离子，进一步产生对入侵病原体（例如烟曲霉）有毒的活性氧 (ROS)。多形核细胞（PMN）利用非氧化和氧化机制来清除肺部的这种真菌。氧化机制主要取决于NOX2的正确组装和功能。我们通过双指数 NAD(P)H 荧光寿命成像 (FLIM) 首次鉴定了参与此类氧化机制的 NAD(P)H 依赖性酶。与线粒体酶结合的 NAD(P)H 的 1870 皮秒相比，3670±140 皮秒的特定荧光寿命可能与活化 PMN 中氧化酶结合的 NADPH 相关。由于它在 PMN 中占主导地位，并且由于选择性激活剂和抑制剂的使用，我们坚信这种特定的寿命主要源自 NOX2。我们的实验还揭示了 NOX2 组装的高位点特异性，从而揭示了 ROS 产生的高位点特异性以及这些现象的动态性质。以 NADPH 氧化酶为例，我们证明了基于 NAD(P)H 的 FLIM 在选择性研究酶的细胞功能过程中的潜力。
NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670±140 picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function.