TY - JOUR

T1 - Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography

AU - Latoscha, Andreas

AU - Drexler, David

AU - Witte, Gregor

AU - Tschowri, Natalia

N1 - Funding Information: Research in the Witte lab is funded by DFG Grant GRK1721 and the DFG Priority Program SPP 1879 (Grant WI 3717/3-1). Research in the Tschowri lab is funded by the DFG Emmy Noether Program (Grant TS 325/1-1) and the DFG Priority Program SPP 1879 (Grants TS 325/2-1 and TS 325/2-2).

PY - 2021/1/5

Y1 - 2021/1/5

N2 - 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.

AB - 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.

KW - AtaC

KW - C-di-AMP

KW - DAC

KW - Diadenylate cyclase

KW - DisA

KW - Enzymatic assay

KW - PDE

KW - Phosphodiesterase

UR - http://www.scopus.com/inward/record.url?scp=85116047631&partnerID=8YFLogxK

U2 - 10.21769/bioprotoc.3870

DO - 10.21769/bioprotoc.3870

M3 - Article

VL - 11

JO - Bio-protocol

JF - Bio-protocol

IS - 1

M1 - e3870

ER -