R&D Systems Inc., Minneapolis, Minnesota, United States of America.
PLoS One. 2011;6(8):e23172. doi: 10.1371/journal.pone.0023172. Epub 2011 Aug 11.
Kinases use adenosine-5'-triphosphate (ATP) as the donor substrate and generate adenosine-5'-diphosphate (ADP) as a product. An ADP-based phosphatase-coupled kinase assay is described here. In this assay, CD39L2, a nucleotidase, is added into a kinase reaction to hydrolyze ADP to AMP and phosphate. The phosphate is subsequently detected using malachite green phosphate-detection reagents. As ADP hydrolysis by CD39L2 displays a first-order rate constant, relatively simple equations are derived to calculate the coupling rate and the lagging time of the coupling reaction, allowing one to obtain kinase kinetic parameters without the completion of the coupling reaction. ATP inhibition of CD39L2-catalyzed ADP hydrolysis is also determined for correction of the kinetic data. As examples, human glucokinase, P. chrysogenum APS kinase and human ERK1, kinases specific for sugar, nucleotide and protein respectively, are assayed. To assess the compatibility of the method for high-throughput assays, Z' factors >0.5 are also obtained for the three kinases.
激酶利用腺苷-5'-三磷酸（ATP）作为供体底物，并生成腺苷-5'-二磷酸（ADP）作为产物。本文描述了一种基于 ADP 的磷酸酶偶联激酶测定法。在该测定法中，CD39L2（一种核苷酸酶）被添加到激酶反应中，以将 ADP 水解为 AMP 和磷酸盐。随后使用孔雀绿磷酸盐检测试剂检测磷酸盐。由于 CD39L2 对 ADP 的水解呈一级反应速率常数，因此推导出了相对简单的方程来计算偶联反应的偶联速率和滞后时间，从而可以在不完成偶联反应的情况下获得激酶动力学参数。还确定了 ATP 对 CD39L2 催化的 ADP 水解的抑制作用，以校正动力学数据。例如，对人葡糖激酶、产黄青霉 APS 激酶和人 ERK1（分别针对糖、核苷酸和蛋白质的激酶）进行了测定。为了评估该方法用于高通量测定的兼容性，还获得了这三种激酶的 Z'因子>0.5。
