Identification and characterization of a novel hepatic canalicular ATP diphosphohydrolase.
We have identified and characterized a novel ATP diphosphohydrolase (ATPDase) 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 localized by immunohistochemistry to the bile canalicular domain of hepatocytes and to the luminal side of the renal ductular epithelium. In contrast, ATPDase/cd39 was detected in vascular endothelium and smooth muscle in these organs. We purified the putative ATPDase from liver by immunoaffinity techniques and obtained a heavily glycosylated protein with a molecular mass estimated at 75 kDa. This enzyme hydrolyzed all tri- and diphosphonucleosides but not AMP or diadenosine polyphosphates. There was an absolute requirement for divalent cations (Ca(2+) > Mg(2+)). Biochemical activity was unaffected by sodium azide or other inhibitors of ATPases. Kinetic parameters derived from purified preparations of hepatic ATPDase indicated V(max) of 8.5 units/mg of protein with apparent K(m) of 100 microM for both ATP or ADP as substrates. NH(2)-terminal amino acid sequencing revealed near 50% identity with rat liver lysosomal (Ca(2+)-Mg(2+))-ATPase. The different biochemical properties and localization of the hepatic ATPDase suggest pathophysiological functions that are distinct from the vascular ATPDase/cd39.
Sévigny
J
J
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA. jsevigny@caregroup.harvard.edu
Robson
S C
SC
Waelkens
E
E
Csizmadia
E
E
Smith
R N
RN
Lemmens
R
R
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000251:Adenosine Triphosphatases; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001081:Apyrase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001648:Bile Canaliculi / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002847:Chromatography, Agarose; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008861:Microsomes / Q000378:metabolism; D019810:Sodium Azide / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine; D013997:Time Factors; D014018:Tissue Distribution
D015703:Antigens, CD; D004791:Enzyme Inhibitors; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D019810:Sodium Azide; D000251:Adenosine Triphosphatases; D001081:Apyrase; C067869:CD39 antigen; D000241:Adenosine; D002118:Calcium