Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-8.
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.
Bigonnesse
François
F
Centre de recherche en Rhumatologie et Immunologie, Université Laval, Sainte-Foy, Québec, Canada.
Lévesque
Sébastien A
SA
Kukulski
Filip
F
Lecka
Joanna
J
Robson
Simon C
SC
Fernandes
Maria J G
MJ
Sévigny
Jean
J
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001081:Apyrase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D005091:Exons; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D014162:Transfection
D018076:DNA, Complementary; D000251:Adenosine Triphosphatases; C027201:ectoATPase; C489209:nucleoside triphosphate diphosphohydrolase 8, mouse; D001081:Apyrase