Functional characterization of rat ecto‐ATPase and ecto‐ATP diphosphohydrolase after heterologous expression in CHO cells

The recently cloned ecto‐ATPase and ecto‐apyrase (ecto‐ATP diphosphohydrolase) are plasma‐membrane‐bound enzymes responsible for the extracellular degradation of nucleoside 5′‐triphosphates and nucleoside 5′‐diphosphates. We expressed the rat‐derived enzymes in CHO cells to compare their molecular and functional properties. Sequence‐specific polyclonal antibodies differentiate between the two proteins and reveal identical molecular masses of 70–80 kDa. Both enzymes are stimulated by either Ca 2+ or Mg 2+ and reveal a broad substrate specificity towards purine and pyrimidine nucleotides. Whereas ecto‐apyrase hydrolyzes nucleoside 5′‐diphosphates at a rate ≈ 20–30% lower than nucleoside‐5′‐triphosphates, ecto‐ATPase hydrolyzes nucleoside‐5′‐diphosphates only to a marginal extent. The sensitivity of the two enzymes to the inhibitors of P2 receptors suramin, PPADS and reactive blue differs. Hydrolysis of ATP by ecto‐ATPase leads to the accumulation in the medium of extracellular ADP as an intermediate product, whereas ecto‐apyrase dephosphorylates ATP directly to AMP. Our results suggest that previous data describing extracellular hydrolysis of ATP by a variety of intact cellular systems with unidentified ecto‐nucleotidases may be explained by the coexpression of ecto‐ATPase and ecto‐apyrase.