Expression and Characterization of Human Ecto‐ATPase and Chimeras with CD39 Ecto‐Apyrase

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Abstract
Human ecto‐ATPase (ecto‐nucleoside triphosphate d iphosphohydrolase 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 (eNTPDase1) 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 diand 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.

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