Identification and characterization of a novel hepatic canalicular ATP diphosphohydrolase

We have identified and characterized a novel ATP diphosphohydrolase (ATPDas e) with features of E-type ATPases from porcine liver. Immunoblotting with a specific monoclonal antibody to this ectoenzyme revealed high expression in liver with lesser amounts in kidney and duodenum. This ATPDase was local ized by immunohistochemistry to the bile canalicular domain of hepatocytes and to the luminal side of the renal ductular epithelium, In contrast, ATPD ase/cd39 was detected in vascular endothelium and smooth muscle in these or gans, We purified the putative ATPDase from liver by immunoaffinity techniq ues and obtained a heavily glycosylated protein with a molecular mass estim ated at 75 kDa. This enzyme hydrolyzed all tri- and diphospho-nucleosides b ut not AMP or diadenosine polyphosphates, There was an absolute requirement for divalent cations (Ca2+ > Mg2+). Biochemical activity was unaffected by sodium azide or other inhibitors of ATPases, Kinetic parameters derived fr om purified preparations of hepatic ATPDase indicated V-max of 8.5 units/mg of protein with apparent K-m of 100 mu M for both ATP or ADP as substrates . NH2-terminal amino acid sequencing revealed near 50% identity with rat li ver lysosomal (Ca2+-Mg2+)-ATPase. The different biochemical properties and localization of the hepatic ATPDase suggest pathophysiological functions th at are distinct from the vascular ATPDase/cd39.