Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography

verfasst von

Andreas Latoscha, David Drexler, Gregor Witte, Natalia Tschowri

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 Streptomyces venezuelae. TLC allows quick and easy separation of radioactive-labeled substrates and products, while IEX avoids utilization of potentially hazardous radioactive substrates and can be used as a good substitute if an HPLC system is not available. Unlike in TLC assays, samples cannot be analyzed in parallel by using the IEX assay, thus it is more time consuming.

Organisationseinheit(en)

Institut für Mikrobiologie

Externe Organisation(en)

Humboldt-Universität zu Berlin (HU Berlin)
Ludwig-Maximilians-Universität München (LMU)

Typ

Artikel

Journal

Bio-protocol

Band

11

Publikationsdatum

05.01.2021

Publikationsstatus

Veröffentlicht

Peer-reviewed

Ja

ASJC Scopus Sachgebiete

Biochemie, Genetik und Molekularbiologie (insg.), Immunologie und Mikrobiologie (insg.), Neurowissenschaften (insg.), Pflanzenkunde

Elektronische Version(en)

https://doi.org/10.21769/bioprotoc.3870 (Zugang: Geschlossen)