Abstract
Ecto-nucleoside-triphosphate diphosphohydrolase-6 (eNTPDase6(1), also known as CD39L2) cDNA was expressed in mammalian COS-1 cells and characterized using nucleotidase assays as well as size exclusion, anion exchange, and cation exchange chromatography. The deduced amino acid sequence of eNTPDase6 is more homologous with the soluble E-type ATPase, eNTPDase5, than other E-type ATPases, suggesting it may also be soluble. To test this possibility, both the cell membranes and the growth media from eNTPDase6-transfected COS-1 cells were assayed for nucleotidase activities. Activity was found in both the membranes and the media. Soluble eNTPDase6 preferentially exhibits nucleoside diphosphatase activity, which is dependent on the presence of divalent cations. Western blot analysis of eNTPDase6 treated with PNGase-F indicated both soluble and membrane-bound forms are glycosylated. However, unlike some membrane-bound ecto-nucleotidases, the eNTPDase6 activity was not specifically inhibited by deglycosylation with peptide N-glycosidase F. Soluble eNTPDase6 hydrolyzed nucleoside triphosphates poorly and nucleoside monophosphates not at all. Analysis of the relative rates of hydrolysis of nucleoside diphosphates (GDP = IDP > UDP > CDP >> ADP) suggests that soluble eNTPDase6 is a diphosphatase most likely not involved in regulation of ADP levels important for circulatory hemostasis.
Highlights
Nucleoside triphosphate diphosphohydrolases (NTPDases)1 are enzymes characterized by their ability to hydrolyze nucleoside tri- and diphosphates
Nucleotidase activity of both the soluble and membrane-bound forms of eNTPDase6 GDPase activities were examined for cation dependence, because this is a defining characteristic for members of the eNTPDase family [1]
This paper describes the first expression of the soluble human nucleotidase eNTPDase6 (CD39L2), and for the first time, the characterization of both soluble and membrane-bound forms of the eNTPDase6
Summary
Materials—Epicurian coli ultracompetent bacteria were purchased from Stratagene. Plasmid purification kits were purchased from Qiagen, Inc. Size Exclusion and Ion Exchange Chromatography—Concentrated culture media from transfected COS cells was applied to a Sephacryl S-200 column equilibrated in 20 mM MOPS, 5 mM MgCl2, pH 7.4. PNGase-F Treatment—For monitoring the effect of deglycosylation on enzyme activity under non-denaturing conditions, samples of crude media (0.2 mg/ml total protein), containing soluble eNTPDase, and, separately, membrane-bound eNTPDase (1.6 mg/ml) were diluted to 0.1 mg/ml in deglycosylation buffer consisting of 30 mM MOPS, 2 mM EDTA, 0.02% saponin, pH 7.2. After various incubation times at 37 °C, 10-l aliquots were removed from the reaction tubes and diluted with nucleotidase assay buffer (20 mM Tris, 1 mM EGTA, 7 mM MgCl2, 100 mM KCl, pH 7.0), and used to determine GDPase activity as described above. All activities are expressed in units of micromoles of Pi/ mg/h, have been COS background-corrected, and are represented as the average of three separate transfections Ϯ S.E
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