Abstract
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.
Highlights
Ectonucleotidases are ubiquitous nucleotide metabolizing enzymes expressed on plasma membranes with externally orientated active sites [1]
Ectonucleotidases at the hepatic sinusoid and bile canalicular membrane may play a key role in the critical regulation of nucleotide and nucleoside trafficking and turnover in the liver, in certain extrahepatic tissues and in bile or plasma [15]
In this study we have identified the hATPDase as a 75 kDa glycoprotein decorated with several N-linked oligosaccharides
Summary
Ectonucleotidases are ubiquitous nucleotide metabolizing enzymes expressed on plasma membranes with externally orientated active sites [1]. The biological function of these enzymes may relate to the regulation of extracellular concentrations of nucleotides (e.g. ATP, ADP, UTP) and nucleosides (e.g. adenosine) [2, 3, 6]. Extracellular ATP and ADP are involved in neurotransmission, nociception, control of secretion from a variety of endocrine and exocrine glands, smooth muscle tone, and vascular hemostasis [8] Such nucleotides and nucleosides act via purinoceptors. Ectonucleotidases at the hepatic sinusoid and bile canalicular membrane may play a key role in the critical regulation of nucleotide and nucleoside trafficking and turnover in the liver, in certain extrahepatic tissues and in bile or plasma [15]. The ectophosphodiesterase family member PC-1 is expressed on the basolateral membrane of hepatocytes, while a closely related protein termed B10 is predominantly canalicular in distribution [20, 21]
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