Mondal Subhanjan, Hsiao Kevin, Goueli Said A
1 Research and Development , Promega Corporation, Madison, Wisconsin.
2 Department of Pathology and Lab Medicine, University of Wisconsin School of Medicine and Public Health , Madison, Wisconsin.
Assay Drug Dev Technol. 2017 Oct/Nov;15(7):330-341. doi: 10.1089/adt.2017.815.
Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are dysregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. In this study, we demonstrate the versatility of an enzyme-coupled assay that quantifies the amount of AMP produced by any enzymatic reaction regardless of its substrates. We successfully implemented it to enzyme reactions that use adenosine triphosphate (ATP) as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP; so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP-generating enzymes that do not require ATP as substrate, including phosphodiesterases (cyclic adenosine monophosphate) and Escherichia coli DNA ligases (nicotinamide adenine dinucleotide [NAD]). In a further elaboration of the AMP-Glo platform, we coupled it to E. coli DNA ligase, enabling measurement of NAD and enzymes that use NAD like monoadenosine and polyadenosine diphosphate-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system, we can measure the activities of sulfotransferases. Thus, by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP-Glo assay.
单磷酸腺苷（AMP）是调节能量稳态和信号转导的关键细胞代谢物。AMP也是各种酶促反应的产物，其中许多在疾病状态下会失调。因此，监测这些酶的活性是开发这些酶调节剂的主要目标。在本研究中，我们展示了一种酶联测定法的多功能性，该方法可量化任何酶促反应产生的AMP量，而不论其底物是什么。我们通过精心设计的策略，即去除残留的三磷酸腺苷（ATP）并将产生的AMP转化为ATP，成功地将其应用于以ATP为底物的酶反应（氨酰tRNA合成酶和DNA连接酶）；这样就可以使用荧光素酶/荧光素进行检测并产生光。我们还测试了该测定法以测量不需要ATP作为底物的产生AMP的酶的活性，包括磷酸二酯酶（环磷酸腺苷）和大肠杆菌DNA连接酶（烟酰胺腺嘌呤二核苷酸[NAD]）。在对AMP-Glo平台的进一步改进中，我们将其与大肠杆菌DNA连接酶偶联，从而能够测量NAD以及使用NAD的酶，如单腺苷和多腺苷二磷酸核糖基转移酶。磺基转移酶使用3'-磷酸腺苷-5'-磷酸硫酸酯作为通用的磺基供体，磷酸腺苷-5'-磷酸（PAP）是通用产物。PAP可以通过高尔基体驻留的PAP特异性磷酸酶IMPAD1转化为AMP来进行定量。通过将IMPAD1与AMP-Glo系统偶联，我们可以测量磺基转移酶的活性。因此，通过利用各种细胞代谢物生化酶促转化为AMP的组合，我们能够证明AMP-Glo测定法的多功能性。
