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MediCity Research Laboratory, University of Turku and National Public Health Institute, FIN-20520, Turku, Finland
1Correspondence: MediCity Research Laboratory, University of Turku, Tykistökatu 6A, FIN-20520, Turku, Finland. E-mail: gennady.yegutkin{at}utu.fi
Cell surface ecto-nucleotidases are considered the major effector system
for inactivation of extracellular adenine nucleotides, whereas the
alternative possibility of ATP synthesis has received little attention.
Using a TLC assay, we investigated the main exchange activities of
3H-labeled adenine nucleotides on the cultured human
umbilical vein endothelial cells. Stepwise nucleotide degradation to
adenosine occurred when a particular nucleotide was present alone,
whereas combined cell treatment with ATP and either
[3H]AMP or [3H]ADP caused unexpected
phosphorylation of 3H-nucleotides via the backward
reactions AMP 
ADP ATP. The following two groups of
nucleotide-converting ecto-enzymes were identified based on inhibition
and substrate specificity studies: 1)
ecto-nucleotidases, ATP-diphosphohydrolase, and 5'-nucleotidase;
2) ecto-nucleotide kinases, adenylate kinase, and
nucleoside diphosphate kinase. Ecto-nucleoside diphosphate kinase
possessed the highest activity, as revealed by comparative kinetic
analysis, and was capable of using both adenine and nonadenine
nucleotides as phosphate donors and acceptors. The transphosphorylation
mechanism was confirmed by direct transfer of the 
-phosphate from
[-32P]ATP to AMP or nucleoside diphosphates and by
measurement of extracellular ATP synthesis using luciferin-luciferase
luminometry. The data demonstrate the coexistence of opposite,
ATP-consuming and ATP-generating, pathways on the cell surface and
provide a novel mechanism for regulating the duration and magnitude of
purinergic signaling in the vasculature.—Yegutkin, G. G.,
Henttinen, T., Jalkanen, S. Extracellular ATP formation on vascular
endothelial cells is mediated by ecto-nucleotide kinase activities via
phosphotransfer reactions.
Key Words: adenine nucleotides • ecto-enzymes • hydrolysis • interconversion • vasculature
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