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 ...
|
|
Monitoring the Targeting of Cathepsin D to the Lysosome by Metabolic Labeling and Pulse-chase Analysis
|
Author:
Date:
2017-11-05
[Abstract] Mannose 6-phosphate receptors function can be studied in living cells by investigating alterations in processing and secretion of their ligand Cathepsin D. The assay described here is well established in the literature and comprises the metabolic labeling of newly synthesized proteins with [35S] methionine-cysteine in HeLa cells to monitor Cathepsin D processing through secretory pathway and secretion using immunoprecipitation, SDS-PAGE and fluorography.
[摘要] 通过研究其配体组织蛋白酶D的加工和分泌的改变,可以在活细胞中研究甘露糖-6-磷酸受体功能。在此描述的测定在文献中已经很好地确定,并且包括新合成的蛋白质的代谢标记,其中[35 [S]甲硫氨酸半胱氨酸,以监测组织蛋白酶D处理,通过分泌途径和分泌使用免疫沉淀,SDS-PAGE和荧光。
【背景】组织蛋白酶d(CATD)是一种溶酶体天冬氨酸蛋白酶由甘露糖-6-磷酸受体(M6PRs)排序在哺乳动物细胞中该传输它从反面高尔基体网络到内涵体/溶酶体(戈什等人,2003 )。将CatD合成为前体蛋白(〜52kDa),其在溶酶体中被切割以产生中间体(〜48kDa)或成熟的溶酶体形式(〜34kDa)。微量的前体蛋白质也从生物合成途径分泌(Benes et al。,2008)。 catD的丰度可以使用几种方法来确定,例如基于免疫荧光的染色(Poole等人,1972),蛋白质印迹,荧光活性测定(Bewley等人, (Hirst等人,2009; Kametaka等人,2007; Tavares等人,2011)或用脉冲追踪分析进行代谢标记(Hirst等人,2009; Kametaka等人, ...
|
|