Development and Validation of a Transcreener Assay for Detection of AMP- and GMP-Producing Enzymes
Development and Validation of a Transcreener Assay for Detection of AMP- and GMP-Producing Enzymes
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DOI：
10.1089/adt.2009.0254
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发表时间：
2010-06-01
影响因子：
1.8
通讯作者：
Lowery, Robert G.
中科院分区：
文献类型：
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作者：
Staeben, Matt;Kleman-Leyer, Karen M.;Lowery, Robert G.
筛选产生AMP和gmp的酶，如磷酸二酯酶（PDEs）、连接酶和合成酶，将通过直接检测未修饰的单磷酸核苷的能力而简化。为了满足这一需求，我们开发了识别AMP和GMP的多克隆和单克隆抗体，具有纳摩尔灵敏度和高选择性，而不是相应的三磷酸和3',5'-环单磷酸核苷酸，这些核苷酸作为这些类别中许多酶的底物。其中一种抗体用于开发具有远红色荧光偏振（FP）读数的Transcreener (R) AMP/GMP测定。该多克隆抗体具有极高的选择性，ATP/AMP的IC（50）比为6000，cAMP/AMP的IC（50）比为3810，cGMP/GMP的IC（50）比为6970。模拟cAMP、cGMP和ATP酶转化为相应的单磷酸盐的标准曲线在10%转化率下的Z值为>0.85。在配药前和配药后，实验试剂在室温下24小时都是稳定的。Transcreener AMP/GMP FP法用于检测cgmp和camp依赖性PDEs 4A1A、3A和9A2以及atp依赖性连接酶、乙酰辅酶a合成酶和泛素活化酶（UBE1）。在测试的所有酶的进展曲线的线性部分观察到>100 mP的变化，并且PDE亚型表现出预期的底物和抑制剂选择性。这些研究表明，对多种产生AMP和GMP的酶进行直接免疫检测是一种灵活、可靠的酶分析方法。此外，它消除了其他方法的许多缺点，包括需要荧光标记的底物，底物耗尽测定中固有的低信号背景，以及与偶联酶干扰的可能性。
Screening of AMP- and GMP-producing enzymes such as phosphodiesterases (PDEs), ligases, and synthetases would be simplified by the ability to directly detect unmodified nucleoside monophosphates. To address this need, we developed polyclonal and monoclonal antibodies that recognize AMP and GMP with nanomolar sensitivity and high selectivity vs. the corresponding triphosphate and 3', 5'-cyclic monophosphate nucleotides that serve as substrates for many enzymes in these classes. One of these antibodies was used to develop a Transcreener (R) AMP/GMP assay with a far red fluorescence polarization (FP) readout. This polyclonal antibody exhibited extremely high selectivity, with IC(50) ratios of 6,000 for ATP/AMP, 3,810 for cAMP/AMP, and 6,970 for cGMP/GMP. Standard curves mimicking enzymatic conversion of cAMP, cGMP, and ATP to the corresponding monophosphates yielded Z' values of >0.85 at 10% conversion. The assay reagents were shown to be stable for 24 h at room temperature, both before and after dispensing. The Transcreener AMP/GMP FP assay was used for enzymatic detection of cGMP-and cAMP-dependent PDEs 4A1A, 3A, and 9A2 and ATP-dependent ligases, acetyl CoA synthetase, and ubiquitin-activating enzyme (UBE1). Shifts of >100 mP were observed in the linear part of the progress curves for all enzymes tested, and the PDE isoforms exhibited the expected substrate and inhibitor selectivity. These studies demonstrate that direct immunodetection of AMP and GMP is a flexible, robust enzyme assay method for diverse AMP- and GMP-producing enzymes. Moreover, it eliminates many of the shortcomings of other methods including the need for fluorescently labeled substrates, the low signal: background inherent in substrate depletion assays, and the potential for interference with coupling enzymes.