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A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile
Published on: November 3, 2018
Dinucleotide polyphosphates contribute to purinergic signalling via inhibition of adenylate kinase activity
Gennady G Yegutkin1, Joachim Jankowski, Sirpa Jalkanen
1MediCity Research Laboratory, University of Turku and National Public Health Institute, Tykistokatu 6A, Turku, 20520 Finland.
Abstract:
Dinucleoside polyphosphates are well described as direct vasoconstrictors and as mediators with strong proliferative properties, however, less is known about their effects on nucleotide-converting pathways. Therefore, the present study investigates the effects of Ap(4)A (diadenosine tetraphosphate), Up(4)A (uridine adenosine tetraphosphate) and Ap(5)A (diadenosine pentaphosphate) and the non-selective P2 antagonist suramin on human serum and endothelial nucleotide-converting enzymes. Human serum and HUVECs (human umbilical vein endothelial cells) were pretreated with various concentrations of dinucleotide polyphosphates and suramin. Adenylate kinase and NDP kinase activities were then quantified radiochemically by TLC analysis of the ATP-induced conversion of [(3)H]AMP and [(3)H]ADP into [(3)H]ADP/ATP and [(3)H]ATP respectively. Endothelial NTPDase (nucleoside triphosphate diphosphohydrolase) activity was additionally determined using [(3)H]ADP and [(3)H]ATP as preferred substrates. Dinucleoside polyphosphates and suramin have an inhibitory effect on the serum adenylate kinase [pIC(50) values (-log IC(50)): Ap(4)A, 4.67+/-0.03; Up(4)A, 3.70+/-0.10; Ap(5)A, 6.31+/-0.03; suramin, 3.74+/-0.07], as well as on endothelial adenylate kinase (pIC(50) values: Ap(4)A, 4.17+/-0.07; Up(4)A, 2.94+/-0.02; Ap(5)A, 5.97+/-0.04; suramin, 4.23+/-0.07), but no significant effects on serum NDP kinase, emphasizing the selectivity of these inhibitors. Furthermore, Ap(4)A, Up(4)A, Ap(5)A and suramin progressively inhibited the rates of [(3)H]ADP (pIC(50) values: Ap(4)A, 3.38+/-0.09; Up(4)A, 2.78+/-0.06; Ap(5)A, 4.42+/-0.11; suramin, 4.10+/-0.07) and [(3)H]ATP (pIC(50) values: Ap(4)A, 3.06+/-0.06; Ap(5)A, 3.05+/-0.12; suramin, 4.14+/-0.05) hydrolyses by cultured HUVECs. Up(4)A has no significant effect on the endothelial NTPDase activity. Although the half-lives for Ap(4)A, Up(4)A and Ap(5)A in serum are comparable with the incubation times of the assays used in the present study, secondary effects of the dinucleotide metabolites are not prominent for these inhibitory effects, since the concentration of metabolites formed are relatively insignificant compared with the 800 mumol/l ATP added as a phosphate donor in the adenylate kinase and NDP kinase assays. This comparative competitive study suggests that Ap(4)A and Ap(5)A contribute to the purinergic responses via inhibition of adenylate-kinase-mediated conversion of endogenous ADP, whereas Up(4)A most likely mediates its vasoregulatory effects via direct binding-mediated mechanisms.
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