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 Ca2+ or Mg2+ and reveal a broad substrate specificity towards purine and pyrimidine nucleotides. Whereas ecto-apyrase hydrolyzes nucleoside 5'-diphosphates at a rate approximately 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.