Determination of diadenosine tetraphosphate in biological material by high pressure liquid chromatography

Abstract

A method for determination of diadenosine tetraphosphate, Ap4A, by high pressure liquid chromatography has been developed. The free nucleotides were extracted from biological material with I 0 % trichloroacetic acid, which subsequently was removed with n-trioctylamine dissolved in freon, the aquous phase containing the nucleotides was lyophilized. The lyophilized material was dissolved in 0. I M-NaHCO3, digested with alkaline phosphatase, injected into a strong anion exchange column without adjustment of pH and chromatographed isocratically with a KH2PO4-KCI buffer at pH 4.3. The enzymatic digestion of the cellular nucleoside mono-, di-, and triphosphates was followed and found to be completed in two hours, while Ap4A was observed not to be cleaved during this period. Amounts as small as 5-10 picomoles of Ap4A were determined quantitatively by the method described.

Diadenosine 5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum. Substrate specificity

“…They also reported a reversed-phase HPLC system that separated Ap4A from monoadenine nucleotides. Plesner & Ottesen (1980) detected Ap4A in extracts from Tetrahymena by HPLC on an anion-exchange column isocratically eluted with a chloride-containing buffer. HPLC of diguanosine polyphosphates and diadenosine polyphosphates other than Ap4A has not been reported.…”

Data_Sheet_1.PDF

“…• Actual function as second messenger currently unclear • In bacteria, both are the degradation product of 3 ,5 -c-di-GMP and 3 ,5 -c-di-AMP, respectively (Rao et al, 2010;Stelitano et al, 2013) • Both have the capability to bind to some targets which are regularly binding the respective, unhydrolyzed c-di-NMP (Smith et al, 2012;Stelitano et al, 2013;Bowman et al, 2016;Kuipers et al, 2016) • As a nanoRNA, both have the potential to affect gene transcription on the level of transcription initiation (Goldman et al, 2011;Nickels and Dove, 2011) Ap 4 A In a multitude of uni-and multicellular species (Plesner and Ottesen, 1980;Flodgaard and Klenow, 1982;Garrison and Barnes, 1984;McLennan and Prescott, 1984) • Potential stress signaling related alarmone (Brevet et al, 1985;Baltzinger et al, 1986;Coste et al, 1987;Garrison et al, 1989) • In metazoa: regulatory effects on the cardiovascular and immune system and neuronal signal transduction (Vahlensieck et al, 1999;Miras-Portugal et al, 2003;Lee et al, 2004) Synthesizing and hydrolyzing enzymes present in various phyla (Ferguson et al, 2020) • Potential stress signaling related alarmone (Lee et al, 1983;Pálfi et al, 1991;Kimura et al, 2017) • Cellular development (Nishimura et al, 1997;Kimura et al, 2017) • Biofilm formation (Monds et al, 2010) Oligo-nucleotide-based signaling molecules cOA According to current knowledge absent in eukaryotes In species utilizing a type III CRISPR system (Kazlauskiene et al, 2017;Niewoehner et al, 2017) • Produced upon presence of invader RNA (Kazlauskiene et al, 2017;Niewoehner et al, 2017) • Activate effectors leading t...…”