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

authored by

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.

Organisation(s)

Institute of Microbiology

External Organisation(s)

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

Type

Article

Journal

Bio-protocol

Volume

11

Publication date

05.01.2021

Publication status

Published

Peer reviewed

Yes

ASJC Scopus subject areas

Biochemistry, Genetics and Molecular Biology(all), Immunology and Microbiology(all), Neuroscience(all), Plant Science

Electronic version(s)

https://doi.org/10.21769/bioprotoc.3870 (Access: Closed)