Expression and characterization of human ecto-ATPase and chimeras with CD39 ecto-apyrase.
Human ecto-ATPase (ecto-nucleoside triphosphate diphosphohydrolase 2 [eNTPDase2], also known as CD39L1) has been expressed and characterized in COS cells. It exhibits some unusual enzymology that is similar to a few members of this class of proteins but different from the majority of the family members. Hydrolysis of ATP by human ecto-ATPase is nonlinear with time, and its activity is stimulated/stabilized by both the lectin concanavalin A and the chemical cross-linking agent disuccinimidyl suberate. Like other members of the eNTPDase family, the human ecto-ATPase is a tetramer, the activity of which depends on its glycosylation. Chimeras of this protein with human CD39 (eNTPDasel) were constructed to test the hypothesis that the N-terminal half of these proteins regulates nucleotide specificity. The two chimeras generated demonstrated that the N-terminal half of these proteins is crucial for determining the relative activities of the nucleoside di- and triphosphatases. Chemical cross-linking of the two chimeras suggests that disuccinimidyl suberate interacts with the C-terminal half of ecto-ATPase in a manner that results in an increase of activity for both the ecto-ATPase and the ecto-apyrase/ecto-ATPase chimera.
Hicks-Berger
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Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Ohio 45267-0575, USA.
Kirley
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D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015703:Antigens, CD / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001081:Apyrase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D003208:Concanavalin A / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D013388:Succinimides / Q000494:pharmacology
D015703:Antigens, CD; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D013388:Succinimides; D003208:Concanavalin A; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase; C067869:CD39 antigen; D004072:Digitonin; C019358:disuccinimidyl suberate