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 diphosphohydrola se) are plasma-membrane-bound enzymes responsible for the extracellular deg radation of nucleoside 5'-triphosphates and nucleoside 5'-diphosphates. We expressed the rat-derived enzymes in CHO cells to compare their molecular a nd functional properties. Sequence-specific polyclonal antibodies different iate between the two proteins and reveal identical molecular masses of 70-8 0 kDa. Both enzymes are stimulated by either Ca2+ or Mg2+ and reveal a broa d substrate specificity towards purine and pyrimidine nucleotides. Whereas ecto-apyrase hydrolyzes nucleoside 5'-diphosphates at a rate approximate to 20-30% lower than nucleoside-5'-triphosphates, ecto-ATPase hydrolyzes nucl eoside-5'-diphosphates only to a marginal extent. The sensitivity of the tw o enzymes to the inhibitors of P2 receptors suramin, PPADS and reactive blu e differs. Hydrolysis of ATP by ecto-ATPase leads to the accumulation in th e medium of extracellular ADP as an intermediate product, whereas ecto-apyr ase dephosphorylates ATP directly to AMP. Our results suggest that previous data describing extracellular hydrolysis of ATP by a variety of intact cel lular systems with unidentified ecto-nucleotidases may be explained by the coexpression of ecto-ATPase and ecto-apyrase.