| Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography
|
|
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 ...
|
|
|
| Aspergillus terreus Infection of Fruits and Terrein Quantification by HPLC Analysis
|
|
Author:
Date:
2016-06-20
[Abstract] The opportunistic fungal human and plant pathogen Aspergillus terreus (A. terreus) can be isolated from sea water, soil or decaying organic matter such as rotting leaves and fruits. While growing on fruits A. terreus produces secondary metabolites such as terrein, which may ease its penetration into plant tissues. In addition, biological activities of terrein may support competition against other microorganisms. In summary, terrein is a small polyketide that reduces germination of seedlings, induces lesions on fruit surfaces but also shows moderate antifungal activity. With this manuscript we provide a fruit infection protocol with Aspergillus terreus with subsequent determination of terrein production rates on infected fruits using an HPLC-based ...
[摘要] 机体真菌人和植物病原菌土曲霉（A. terreus）可以从海水，土壤或腐烂的有机物质如腐烂的叶子和果实中分离出来。 当在水果上生长时，A. terreus产生次生代谢物如土曲杆菌，这可以缓解其渗透到植物组织中。 此外，土耳特蛋白的生物活性可能支持与其他微生物的竞争。 总而言之，土豆蛋白是一种减少幼苗发芽的小聚酮，诱发水果表面的病变，但也显示出中等的抗真菌活性。 使用这份手稿，我们提供了一种水果感染方案与土曲霉，随后使用基于HPLC的量化方法测定感染果实的土豆油生产率。
|
|
|