Analysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with N'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.

As second messengers, the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) play an essential role in intracellular signaling. Recent data suggest that the cyclic pyrimidine nucleotides cytidine 3',5'-c...

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Autores principales:	Daniel Reinecke, Frank Schwede, Hans-Gottfried Genieser, Roland Seifert
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5e722d6db7794d5e9e93f0bfa3bea681
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spelling	oai:doaj.org-article:5e722d6db7794d5e9e93f0bfa3bea6812021-11-18T08:01:31ZAnalysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with N'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.1932-620310.1371/journal.pone.0054158https://doaj.org/article/5e722d6db7794d5e9e93f0bfa3bea6812013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23342095/?tool=EBIhttps://doaj.org/toc/1932-6203As second messengers, the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) play an essential role in intracellular signaling. Recent data suggest that the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP) and uridine 3',5'-cyclic monophosphate (cUMP) also act as second messengers. Hydrolysis by phosphodiesterases (PDEs) is the most important degradation mechanism for cAMP and cGMP. Elimination of cUMP and cCMP is not completely understood, though. We have shown that human PDEs hydrolyze not only cAMP and cGMP but also cyclic pyrimidine nucleotides, indicating that these enzymes may be important for termination of cCMP- and cUMP effects as well. However, these findings were acquired using a rather expensive HPLC/mass spectrometry assay, the technical requirements of which are available only to few laboratories. N'-Methylanthraniloyl-(MANT-)labeled nucleotides are endogenously fluorescent and suitable tools to study diverse protein/nucleotide interactions. In the present study, we report the synthesis of new MANT-substituted cyclic purine- and pyrimidine nucleotides that are appropriate to analyze substrate specificity and kinetics of PDEs with more moderate technical requirements. MANT-labeled nucleoside 3',5'-cyclic monophosphates (MANT-cNMPs) are shown to be substrates of various human PDEs and to undergo a significant change in fluorescence upon cleavage, thus allowing direct, quantitative and continuous determination of hydrolysis via fluorescence detection. As substrates of several PDEs, MANT-cNMPs show similar kinetics to native nucleotides, with some exceptions. Finally, they are shown to be also appropriate tools for PDE inhibitor studies.Daniel ReineckeFrank SchwedeHans-Gottfried GenieserRoland SeifertPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 1, p e54158 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Daniel Reinecke Frank Schwede Hans-Gottfried Genieser Roland Seifert Analysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with N'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.
description	As second messengers, the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) play an essential role in intracellular signaling. Recent data suggest that the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP) and uridine 3',5'-cyclic monophosphate (cUMP) also act as second messengers. Hydrolysis by phosphodiesterases (PDEs) is the most important degradation mechanism for cAMP and cGMP. Elimination of cUMP and cCMP is not completely understood, though. We have shown that human PDEs hydrolyze not only cAMP and cGMP but also cyclic pyrimidine nucleotides, indicating that these enzymes may be important for termination of cCMP- and cUMP effects as well. However, these findings were acquired using a rather expensive HPLC/mass spectrometry assay, the technical requirements of which are available only to few laboratories. N'-Methylanthraniloyl-(MANT-)labeled nucleotides are endogenously fluorescent and suitable tools to study diverse protein/nucleotide interactions. In the present study, we report the synthesis of new MANT-substituted cyclic purine- and pyrimidine nucleotides that are appropriate to analyze substrate specificity and kinetics of PDEs with more moderate technical requirements. MANT-labeled nucleoside 3',5'-cyclic monophosphates (MANT-cNMPs) are shown to be substrates of various human PDEs and to undergo a significant change in fluorescence upon cleavage, thus allowing direct, quantitative and continuous determination of hydrolysis via fluorescence detection. As substrates of several PDEs, MANT-cNMPs show similar kinetics to native nucleotides, with some exceptions. Finally, they are shown to be also appropriate tools for PDE inhibitor studies.
format	article
author	Daniel Reinecke Frank Schwede Hans-Gottfried Genieser Roland Seifert
author_facet	Daniel Reinecke Frank Schwede Hans-Gottfried Genieser Roland Seifert
author_sort	Daniel Reinecke
title	Analysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with N'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.
title_short	Analysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with N'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.
title_full	Analysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with N'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.
title_fullStr	Analysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with N'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.
title_full_unstemmed	Analysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with N'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.
title_sort	analysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with n'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/5e722d6db7794d5e9e93f0bfa3bea681
work_keys_str_mv	AT danielreinecke analysisofsubstratespecificityandkineticsofcyclicnucleotidephosphodiesteraseswithnmethylanthraniloylsubstitutedpurineandpyrimidine35cyclicnucleotidesbyfluorescencespectrometry AT frankschwede analysisofsubstratespecificityandkineticsofcyclicnucleotidephosphodiesteraseswithnmethylanthraniloylsubstitutedpurineandpyrimidine35cyclicnucleotidesbyfluorescencespectrometry AT hansgottfriedgenieser analysisofsubstratespecificityandkineticsofcyclicnucleotidephosphodiesteraseswithnmethylanthraniloylsubstitutedpurineandpyrimidine35cyclicnucleotidesbyfluorescencespectrometry AT rolandseifert analysisofsubstratespecificityandkineticsofcyclicnucleotidephosphodiesteraseswithnmethylanthraniloylsubstitutedpurineandpyrimidine35cyclicnucleotidesbyfluorescencespectrometry
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Analysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with N'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.

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