Cloning and Characterization of Mouse Nucleoside Triphosphate Diphosphohydrolase-8^,

Abstract

A novel mammalian plasma membrane bound nucleoside triphosphate diphosphohydrolase (NTPDase), named NTPDase8, has been cloned and characterized. Analysis of cDNA reveals an open reading frame of 1491 base pairs encoding a protein of 497 amino acid residues with an estimated molecular mass of 54650 Da and a predicted isoelectric point of 5.94. In a mouse, the genomic sequence is located on chromosome 2A3 and is comprised of 10 exons. The deduced amino acid sequence reveals eight putative N-glycosylation sites, two transmembrane domains, five apyrase-conserved regions, and 20-50% amino acid identity with other mammalian NTPDases. mRNA expression was detected in liver, jejunum, and kidney. Both intact cells and crude cell lysates from COS-7 cells expressing NTPDase8 hydrolyzed P2 receptor agonists, namely, ATP, ADP, UTP, and UDP, but did not hydrolyze AMP. There was an absolute requirement for divalent cations for the catalytic activity (Ca(2+) > Mg(2+)) with an optimal pH between 5.5 and 8.0 for ATP and 6.4 for ADP hydrolysis. Kinetic parameters derived from analysis of crude cell lysates showed that the enzyme had lower apparent K(m) values for adenine nucleotides and for triphosphonucleosides (K(m,app) of 13 microM for ATP, 41 microM for ADP, 47 microM for UTP, and 171 microM for UDP). Hydrolysis of triphosphonucleosides resulted in a transient accumulation of the corresponding diphosphonucleoside, as expected from the apparent K(m) values. Enzymatic properties of NTPDase8 differ from those of other NTPDases suggesting an alternative way to modulate nucleotide levels and consequently P2 receptor activation.

Reduced striatal ecto-nucleotidase activity in schizophrenia patients supports the “adenosine hypothesis”

“…In the present study we did not look at the other two membrane-associated members of the NTPDase family, NTPDase2, and NTPDase8. It is known that in rodents, NTPDase2 is expressed in neural stem cells in the subventricular zone of the lateral ventricles [37], and no ntpdase8 mRNA could be detected by PCR in mouse brain [38].…”

Portal Fibroblasts Regulate the Proliferation of Bile Duct Epithelia via Expression of NTPDase2

“…NTPDase2 is one of a small collection of ecto-nucleotidases of the ecto-nucleoside triphosphate diphosphohydrolase family (39,40). These enzymes all share catalytic activity for nucleotides such as ATP.…”

Ectonucleotidases in the kidney

“…For most of these enzymes, nucleotides derived from other bases (UTP, GTP, TTP, CTP, UDP, GDP, TDP and CDP) can also act as substrates hydrolysis of tri-over diphosphonucleosides, though in these cases the preference is less marked [8,12]. Thus, NTPDases 3 and 8 generate only a transient accumulation of diphosphonucleosides [10,13,14].…”