A scintillation proximity assay for poly(ADP-ribose) polymerase
A scintillation proximity assay for poly(ADP-ribose) polymerase
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DOI：
10.1006/abio.2000.4604
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发表时间：
2000-06-15
影响因子：
2.9
通讯作者：
Zhang, J
中科院分区：
文献类型：
--
作者：
Cheung, A;Zhang, J
多聚腺苷二磷酸核糖聚合酶（Poly（ADP-ribose）polymerase，PARP）是一种广泛存在于真核生物组织中的核蛋白。当被DNA损伤激活时，PARP从NAD合成聚（ADP-核糖）。常规的放射性PARP酶测定需要将聚合物产物与NAD底物分离，这是一个限速步骤，阻碍了大规模化学文库筛选以鉴定新型小分子PARP抑制剂。通过使用生物素化NAD，我们已经开发了PARP的闪烁接近测定（SPA）。我们证明了PARP可以将生物素化的ADP-核糖单元掺入放射性聚（ADP-核糖）聚合物中，其可以直接结合并激发链霉亲和素缀合的闪烁珠。PARP-SPA可以很容易地适应于96孔格式，用于自动高通量筛选PARP抑制剂。(C)北京大学出版社.
Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear protein in most of the eukaryotic tissues. When activated by DNA damage, PARP synthesizes poly(ADP-ribose) from NAD, Conventional radioactive PARP enzyme assay requires the separation of the polymer product from the NAD substrate, a rate-limiting step that hampers large-scale chemical library screening to identify novel small-molecule PARP inhibitors. By using biotinylated NAD, we have developed a scintillation proximity assay (SPA) for PARP. We demonstrated that PARP can incorporate the biotinylated ADP-ribose units into the radioactive poly-(ADP-ribose) polymer, which can directly bind and excite the streptavidin-conjugated scintillation beads. PARP-SPA can be readily adapted to a 96-well format for automatic high-throughput screening for PARP inhibitors. (C) 2000 Academic Press.