Detection of poly(ADP-ribose) polymerase activation in oxidatively stressed cells and tissues using biotinylated NAD substrate
Detection of poly(ADP-ribose) polymerase activation in oxidatively stressed cells and tissues using biotinylated NAD substrate
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DOI：
10.1177/002215540205000110
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发表时间：
2002-01-01
影响因子：
3.2
通讯作者：
Virág, L
中科院分区：
文献类型：
--
作者：
Bakondi, E;Bai, P;Virág, L
聚ADP核糖聚合酶（Poly（ADP-ribose）polymerase，PARP）是一种由DNA损伤激活的核酶。活化的PARP将NAD（+）切割成烟酰胺和（ADP-核糖），并将后者聚合在核受体蛋白上。活性氧和氮中间体对PARP的过度激活代表了各种形式的炎症、休克和再灌注损伤的致病因素。使用一种新的市售底物，6-生物素-17-烟酰胺腺嘌呤二核苷酸（bio-NAD（+）），我们已经开发了三种应用，酶细胞化学，酶组织化学和细胞ELISA，检测氧化应激细胞和组织中PARP的激活。使用新的测定法，我们能够检测J774巨噬细胞中的基础和过氧化氢诱导的PARP活性。我们还观察到，有丝分裂细胞显示显着升高的PARP活性。过氧化氢诱导的PARP激活也可以在野生型腹膜巨噬细胞中检测到，但在PARP缺陷小鼠的巨噬细胞中未检测到。将过氧化氢应用于小鼠皮肤也诱导bio-NAD（+）掺入角质形成细胞核中。用ELISA法检测J774细胞中过氧化氢诱导的PARP激活及其药物PARP抑制剂的抑制作用，与传统的[H-3]-NAD掺入法显示出良好的相关性。bio-NAD（+）检测试剂盒代表了检测PARP激活的灵敏度、特异性和非放射性替代方法。
Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by DNA damage. Activated PARP cleaves NAD(+) into nicotinamide and (ADP-ribose) and polymerizes the latter on nuclear acceptor proteins. Over-activation of PARP by reactive oxygen and nitrogen intermediates represents a pathogenetic factor in various forms of inflammation, shock, and reperfusion injury. Using a novel commercially available substrate, 6-biotin-17-nicotinamide-adenine-dinucleotide (bio-NAD(+)), we have developed three applications, enzyme cytochemistry, enzyme histochemistry, and cell ELISA, to detect the activation of PARP in oxidatively stressed cells and tissues. With the novel assay we were able to detect basal and hydrogen peroxide- induced PARP activity in J774 macrophages. We also observed that mitotic cells display remarkably elevated PARP activity. Hydrogen peroxide-induced PARP activation could also be detected in wild-type peritoneal macrophages but not in macrophages from PARP-deficient mice. Application of hydrogen peroxide to the skin of mice also induced bio-NAD(+) incorporation in the keratinocyte nuclei. Hydrogen peroxide-induced PARP activation and its inhibition by pharmacological PARP inhibitors could be detected in J774 cells with the ELISA assay that showed good correlation with the traditional [H-3]-NAD incorporation method. The bio-NAD(+) assays represent sensitive, specific, and non-radioactive alternatives for detection of PARP activation.