| Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography
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Author:
Date:
2021-01-05
[Abstract] All living cells use cyclic nucleotides as second messengers for signal sensing and transduction. Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is primarily involved in the control of bacterial and euryarcheal osmoadaptation and is produced by diadenylate cyclases from two molecules of ATP. Specific phosphodiesterases hydrolyze c-di-AMP to the linear phosphoadenylate adenosine 5′-pApA or to AMP. Different methods including high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and ion exchange chromatography (IEX) can be used to determine activities of c-di-AMP-synthesizing and degrading enzymes. Here, we describe in detail the TLC and IEX methods adapted for characterization of the diadenylate cyclase DisA and the phosphodiesterase AtaC from ...
[摘要] [摘要]所有活细胞均使用环状核苷酸作为第二信使，以进行信号传感和转导。环状二-3 ' ，5 ' -腺苷一磷酸（C-二-AMP）主要涉及细菌和控制euryarcheal osmoadaptation并且由产生diadenylate环化酶从ATP的两个分子。特定的磷酸二酯酶将c-di-AMP水解为线性磷酸腺苷腺苷5'-pApA或AMP。可以使用包括高效液相色谱（HPLC），薄层色谱（TLC）和离子交换色谱（IEX）在内的各种方法来确定c-di-AMP合成和降解酶的活性。在这里，我们详细描述了TLC和IEX方法适合于表征diadenylate环化酶DISA和磷酸二酯酶ATAC从链霉菌venezuelae 。TLC可以快速，轻松地分离放射性标记的底物和产品，而IEX避免了潜在危险的放射性底物的利用，如果没有HPLC系统，则可以用作良好的替代品。与TLC分析不同，无法使用IEX分析并行分析样品，因此更加耗时。
[背景]环核苷酸第二信使是原核和真核信号通路中的关键分子。环状二-3 ' ，5 ' -腺苷一磷酸（三二- AMP）是一种细菌第二信使与许多重要的功能，如渗压剂动态平衡调节，细胞壁代谢，生物膜的形成，DNA完整，孢子形成，毒力和生长（Fahmi et ...
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| Measuring cAMP Specific Phosphodiesterase Activity: A Two-step Radioassay
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Author:
Date:
2020-04-05
[Abstract] Cyclic nucleotide degrading phosphodiesterase (PDE) enzymes are crucial to the fine tuning of cAMP signaling responses, playing a pivotal role in regulating the temporal and spatial characteristics of discrete cAMP nanodomains and hence the activity of cAMP-effector proteins. As a consequence of orchestrating cAMP homeostasis, dysfunctional PDE activity plays a central role in disease pathogenesis. This highlights the need for developing methods that can be used to further understand PDE function and assess the effectiveness of potentially novel PDE therapeutics. Here we describe such an approach, where PDE activity is indirectly measured through the direct quantification of radioactively tagged cAMP (pmol/min-1/mg-1). This method provides a highly sensitive tool for ...
[摘要] [摘要 ] 环核苷酸降解磷酸二酯酶（PDE）酶对cAMP信号响应的微调至关重要，在调节离散cAMP纳米域的时空特征以及cAMP效应子蛋白的活性中起着关键作用。协调cAMP稳态的结果是，功能异常的PDE活性在疾病发病机理中起着核心作用。这突出了对开发可用于进一步了解PDE功能并评估潜在的新型PDE治疗剂有效性的方法的需求。在这里，我们描述了这种方法，其中PDE活性是通过对放射性标记的cAMP（pmol / min -1 / mg -1 ）。此方法为调查PDE功能提供了高度敏感的工具。
[背景 ] 环状3'，5'-单磷酸腺苷（cAMP）是由腺苷酸环化酶（ACs）合成的普遍表达的第二信使，其充当细胞内信号转导通路的广泛调控者（Hayes and Brunton，1982） ；Beavo 和Brunton，2002年）。磷酸二酯酶（PDE）可精细调节cAMP的体内稳态，从而将cAMP迅速降解为5'AMP（Baillie，2009年）。PDE由11个家族（PDE1-11）组成，跨越100多种同工酶（例如PDE4A1-PDE4A5），均具有快速水解环状核苷酸的能力（Conti和Beavo ...
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