| 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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| Investigating the Assembly Status of the Plastid Encoded Polymerase Using BN-PAGE and Sucrose Gradient Centrifugation
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Author:
Date:
2016-07-20
[Abstract] The plastid encoded polymerase (PEP) represents a major transcription machinery in mature chloroplasts (Liere et al., 2011; Zhelyazkova et al., 2012). The proper assembly of this multi-subunit complex is important for plant growth and development (Pfalz and Pfannschmidt, 2013). The PEP polymerase can be purified from soluble and from membrane-bound (also named transcriptionally active chromosome, TAC) fractions. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) and sucrose gradient sedimentation followed by immunoblot analyses is used to detect the status of the PEP complex assembly.
[摘要] 质体编码聚合酶（PEP）代表成熟叶绿体中的主要转录机制（Liere等人，2011; Zhelyazkova等人，2012）。 这种多亚基复合物的正确装配对于植物生长和发育是重要的（Pfalz和Pfannschmidt，2013）。 PEP聚合酶可以从可溶性和膜结合（也称为转录活性染色体，TAC）级分中纯化。 蓝色使用天然聚丙烯酰胺凝胶电泳（BN-PAGE）和蔗糖梯度沉淀，随后进行免疫印迹分析来检测PEP复合物组装体的状态。
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