Development of a Novel Ectonucleotidase Assay Suitable for High-Throughput Screening
Development of a Novel Ectonucleotidase Assay Suitable for High-Throughput Screening
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DOI：
10.1177/1087057112443987
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发表时间：
2012-08-01
影响因子：
--
通讯作者：
Hollingsworth, Robert
中科院分区：
文献类型：
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作者：
Sachsenmeier, Kris F.;Hay, Carl;Hollingsworth, Robert
5'-外核苷酸酶 (NT5E) 催化单磷酸腺苷转化为腺苷和游离磷酸盐。这种核酸外切酶的作用及其腺苷的产生与免疫功能、血管生成和癌症有关。 NT5E 活性通常通过使用高效液相色谱 (HPLC) 对底物和产物进行色谱定量或通过使用孔雀石绿对游离磷酸盐进行定量来测定。这些方法不适用于 NT5E 活性的稳健筛选测定。 HPLC 不太适合快速有效地检测多个样品，并且孔雀石绿对各种介质和样品缓冲液中常见的含磷酸盐缓冲液高度敏感。这里描述了一种新型高通量核酸酶筛选测定的开发和验证，该测定利用基于荧光素酶的测定试剂 Promega CellTiter-Glo 试剂盒来测量 NT5E 对 AMP 的分解代谢。这种基于多孔板的测定有助于筛选潜在的核酸外切酶拮抗剂，并且不受筛选样品中存在的污染性磷酸盐分子的影响。
5'-Ectonucleotidase (NT5E) catalyzes the conversion of adenosine monophosphate to adenosine and free phosphate. The role of this ectonucleotidase and its production of adenosine are linked with immune function, angiogenesis, and cancer. NT5E activity is typically assayed either by chromatographic quantification of substrates and products using high-performance liquid chromatography (HPLC) or by quantification of free phosphate using malachite green. These methods are not suitable for robust screening assays of NT5E activity. HPLC is not readily suitable for the rapid and efficient assay of multiple samples and malachite green is highly sensitive to the phosphate-containing buffers common in various media and sample buffers. Here the development and validation of a novel high-throughput ectonucleotidase screening assay are described, which makes use of a luciferase-based assay reagent, the Promega CellTiter-Glo kit, to measure the catabolism of AMP by NT5E. This multiwell plate-based assay facilitates the screening of potential ectonucleotidase antagonists and is unaffected by the presence of contaminating phosphate molecules present in screening samples.