Details
| Original language | English |
|---|---|
| Article number | e3870 |
| Journal | Bio-protocol |
| Volume | 11 |
| Issue number | 1 |
| Publication status | Published - 5 Jan 2021 |
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.
Keywords
- AtaC, C-di-AMP, DAC, Diadenylate cyclase, DisA, Enzymatic assay, PDE, Phosphodiesterase
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- General Biochemistry,Genetics and Molecular Biology
- Immunology and Microbiology(all)
- General Immunology and Microbiology
- Neuroscience(all)
- General Neuroscience
- Agricultural and Biological Sciences(all)
- Plant Science
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In: Bio-protocol, Vol. 11, No. 1, e3870, 05.01.2021.
Research output: Contribution to journal › Article › Research › peer review
}
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 -