PDE7A1 hydrolyzes cCMP |
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Authors: | Maike Monzel,Maike Kuhn,Heike Bä hre,Roland Seifert,Erich H. Schneider |
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Affiliation: | 1. Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany;2. Core Facility Mass Spectrometry and Metabolomics, Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany |
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Abstract: | The degradation and biological role of the cyclic pyrimidine nucleotide cCMP is largely elusive. We investigated nucleoside 3′,5′-cyclic monophosphate (cNMP) specificity of six different recombinant phosphodiesterases (PDEs) by using a highly-sensitive HPLC–MS/MS detection method. PDE7A1 was the only enzyme that hydrolyzed significant amounts of cCMP. Enzyme kinetic studies using purified GST-tagged truncated PDE7A1 revealed a cCMP KM value of 135 ± 19 μM. The Vmax for cCMP hydrolysis reached 745 ± 27 nmol/(min mg), which is about 6-fold higher than the corresponding velocity for adenosine 3′,5′-cyclic monophosphate (cAMP) degradation. In summary, PDE7A is a high-speed and low-affinity PDE for cCMP. |
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Keywords: | A549, human lung carcinoma cell line AMP, adenosine 5&prime -monophosphate cAMP, adenosine 3&prime ,5&prime -cyclic monophosphate B103, rat neuroblastoma cells CMP, cytidine 5&prime -monophosphate cCMP, cytidine 3&prime ,5&prime -cyclic monophosphate CyaA, Bordetella pertussis adenylyl cyclase toxin ExoY, P. aeruginosa exotoxin Y GMP, guanosine 5&prime -monophosphate cGMP, guanosine 3&prime ,5&prime -cyclic monophosphate GST, glutathione-S-transferase protein tag HCN, hyperpolarization-activated cyclic nucleotide-gated cation channel HEK-293, human embryonic kidney cell line HIS, hexahistidine tag HPLC&ndash MS/MS, high performance liquid chromatography tandem mass spectrometry HuT 78, human lymphoblast cell line IMP, inosine 5&prime -monophosphate cIMP, inosine 3&prime ,5&prime -cyclic monophosphate MRP5, multidrug resistance-associated protein 5 cNMP, nucleoside 3&prime ,5&prime -cyclic monophosphate PDE, phosphodiesterase PKA, protein kinase A PKG, protein kinase G Sf9, Spodoptera frugiperda insect cell line TMP, thymidine 5&prime -monophosphate cTMP, thymidine 3&prime ,5&prime -cyclic monophosphate UMP, uridine 5&prime -monophosphate cUMP, uridine 3&prime ,5&prime -cyclic monophosphate U-937, human promonocytic cell line XMP, xanthosine 5&prime -monophosphate cXMP, xanthosine 3&prime ,5&prime -cyclic monophosphate |
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