NTPDases - apyrases (ATP diphosphohydrolases; EC 3.6.1.5.) are enzymes capable of hydrolyzing 5'-, di- and triphosphate nucleotides to di- and monophosphate nucleotides and an inorganic phosphate. These enzymes have been detected in plants, vertebrates, insects, parasites, protozoa and yeast, in almost all kinds of tissues and cells. The animals have eight different enzymes encoded by eight different genes. Despite the high similarity of nucleotide and amino acid sequences, NTPDases reveal different substrate specificity. Basic role of NTPDases is regulation of levels of intra- and extracellular nucleotides and nucleosides. They participate in the metabolism of ecto-nucleotides (ATP, ADP), which play a significant role in regulation of blood pressure and platelet aggregation. Disorders in these processes might cause atherosclerosis and myocardial infarction. Aim of the research is to develop a new anticoagulant enzymatic drug based on the nucleotidase activity of the plant NTPDases - apyrases.

The research was conducted with cDNA of A2F2 NTPDase from A. thaliana and E. coli BL21(DE3)Codon+ bacteria. The apyrase gene was cloned into pET-28a plasmide. That vector was used to transform bacteria. The overexpressed protein accumulated in inclusion bodies.

The refolding process by dilution was performed to restore the biological activity. Different contents of the inclusion body proteins were tested to optimize the conditions of refolding with the selected method. The maximum efficiency was achieved with the largest (36 mg/ml) initial concentration of the protein. However, only 7% of the used protein was reactivated. The largest specific activity was obtained for refolding performed with the 1,98 mg/ml protein of inclusion bodies. The active protein was purified with the ion exchange chromatography. Separation with the MonoQ column gave two catalytically active fractions (A and B) differing by the substrate specificity against adenine nucleotides (ATP and ADP). Fraction A was eluted from the column without using KCl. That enzyme hydrolyzed both ATP and ADP. Second fraction, eluted with 0,7 M KCl, degraded only ADP. Activity of both enzymes was stimulated by the presence of Mg²⁺ ions. Electroforesis of both fractions under denaturing conditions revealed the presence of bands with a molecular mass corresponding to the product of the apyrase gene. Separation of these proteins under native conditions revealed, that they have significantly different mobility. Western Blotting confirmed that the refolded and purified proteins are NTPDases.

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1. The influence of E-NTPDase 1 (apyrase EC 3.6.1.5) and adenylate kinase (EC 2.7.4.3) on pig blood's platelet aggregation.
2. Identification of ecto-nucleotidases in human cerebrospinal fluid
3. Application of the enzymatic assay of cholesterol to the examination of cholesterol metabolism in the lysosomal membranes