Biochemical characterization of soluble nucleotide pyrophosphatase/phosphodiesterase activity in rat serum.

Danijela Laketa, Ivana Bjelobaba, Jasmina Savic, Irena Lavrnja, Mirjana Stojiljkovic, Ljubisav Rakic, Nadezda Nedeljkovic

Institute for Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski Trg 3, 11001, Belgrade, Serbia.

Molecular and cellular biochemistry 2010 Jun

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Biochemical properties of nucleotide pyrophosphatase/phosphodiesterase (NPP) in rat serum have been described by assessing its nucleotide phosphodiesterase activity, using p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as a substrate. It was demonstrated that NPP activity shares some typical characteristics described for other soluble NPP, such as divalent cation dependence, strong alkaline pH optimum (pH 10.5), inhibition by glycosaminoglycans, and K (m) for p-Nph-5'-TMP hydrolysis of 61.8 +/- 5.2 microM. In order to characterize the relation between phosphodiesterase and pyrophosphatase activities of NPP, we have analyzed the effects of different natural nucleotides and nucleotide analogs. ATP, ADP, and AMP competitively inhibited p-Nph-5'-TMP hydrolysis with K (i) values ranging 13-43 microM. Nucleotide analogs, alpha,beta-metATP, BzATP, 2-MeSATP, and dialATP behaved as competitive inhibitors, whereas alpha,beta-metADP induced mixed inhibition, with K (i) ranging from 2 to 20 microM. Chromatographic analysis revealed that alpha,beta-metATP, BzATP, and 2-MeSATP were catalytically degraded in the serum, whereas dialATP and alpha,beta-metADP resisted hydrolysis, implying that the former act as substrates and the latter as true competitive inhibitors of serum NPP activity. Since NPP activity is involved in generation, breakdown, and recycling of extracellular adenine nucleotides in the vascular compartment, the results suggest that both hydrolyzable and non-hydrolyzable nucleotide analogs could alter the amplitude and direction of ATP actions and could have potential therapeutic application.

Citation

Danijela Laketa, Ivana Bjelobaba, Jasmina Savic, Irena Lavrnja, Mirjana Stojiljkovic, Ljubisav Rakic, Nadezda Nedeljkovic. Biochemical characterization of soluble nucleotide pyrophosphatase/phosphodiesterase activity in rat serum. Molecular and cellular biochemistry. 2010 Jun;339(1-2):99-106