Cloning, purification, and identification of the liver canalicular ecto-ATPase as NTPDase8
| Autor: | Jonathan A. Dranoff, Sébastien A. Lévesque, Michel Fausther, Julie Pelletier, Joanna Lecka, Filip Kukulski, Jean Sévigny, Herbert Zimmermann |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Physiology
ATPase Molecular Sequence Data Biology Catalysis Article Cell Line Substrate Specificity Rats Sprague-Dawley Antigens CD Cell surface receptor Physiology (medical) Chlorocebus aethiops Extracellular Animals Humans Nucleotide Amino Acid Sequence Cloning Molecular Enzyme Inhibitors Sodium Azide Adenosine Triphosphatases chemistry.chemical_classification Cloning Sequence Homology Amino Acid Hepatology Nucleotides Apyrase Gastroenterology Recombinant Proteins Rats Kinetics Liver chemistry Biochemistry Cell culture COS Cells Hepatocytes biology.protein Nucleoside Deoxycholic Acid |
| Zdroj: | American Journal of Physiology-Gastrointestinal and Liver Physiology. 292:G785-G795 |
| ISSN: | 1522-1547 0193-1857 |
| DOI: | 10.1152/ajpgi.00293.2006 |
| Popis: | Extracellular nucleotides regulate critical liver functions via the activation of specific transmembrane receptors. The hepatic levels of extracellular nucleotides, and therefore the related downstream signaling cascades, are modulated by cell-surface enzymes called ectonucleotidases, including nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2/CD39L1, and ecto-5′-nucleotidase/CD73. The goal of this study was to determine the molecular identity of the canalicular ecto-ATPase/ATPDase that we hypothesized to correspond to the recently cloned NTPDase8. Human and rat NTPDase8 cDNAs were cloned, and the genes were located on chromosome loci 9q34 and 3p13, respectively. The recombinant proteins, expressed in COS-7 and HEK293T cells, were biochemically characterized. NTPDase8 was also purified from rat liver by Triton X-100 solubilization, followed by DEAE, Affigel Blue, and concanavalin A chromatographies. Importantly, NTPDase8 was responsible for the major ectonucleotidase activity in liver. The ion requirement, apparent Kmvalues, nucleotide hydrolysis profile, and preference as well as the resistance to azide were similar for recombinant NTPDase8s and both purified rat NTPDase8 and porcine canalicular ecto-ATPase/ATPDase. The partial NH2-terminal amino acid sequences of all NTPDase8s share high identity with the purified liver canalicular ecto-ATPase/ATPDase. Histochemical analysis showed high ectonucleotidase activities in bile canaliculi and large blood vessels of rat liver, in agreement with the immunolocalization of NTPDase1, 2, and 8 with antibodies developed for this study. No NTPDase3 expression could be detected in liver. In conclusion, NTPDase8 is the canalicular ecto-ATPase/ATPDase and is responsible for the main hepatic NTPDase activity. The canalicular localization of this enzyme suggests its involvement in the regulation of bile secretion and/or nucleoside salvage. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|