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.
Heine
P
P
AK Neurochemie, Biozentrum der J.W. Goethe-Universität, Frankfurt am Main, Germany.
Braun
N
N
Heilbronn
A
A
Zimmermann
H
H
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001081:Apyrase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D002412:Cations; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006868:Hydrolysis; D008969:Molecular Sequence Data; D051381:Rats; D013379:Substrate Specificity
D002412:Cations; D017931:DNA Primers; D004791:Enzyme Inhibitors; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase