Cloning and Characterization of Mouse Nucleoside Triphosphate Diphosphohydrolase-8
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- 作者：Franç ; ois Bigonnesse ; Sé ; bastien A. Lé ; vesque ; Filip Kukulski ; Joanna Lecka ; Simon C. Robson ; Maria J. G. Fernandes ; and Jean Sé ; vigny
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：May 11, 2004
- 年：2004
- 卷：43
- 期：18
- 页码：5511 - 5519
- 全文大小：220K
- 年卷期：v.43,no.18(May 11, 2004)
- ISSN：1520-4995
文摘
A novel mammalian plasma membrane bound nucleoside triphosphate diphosphohydrolase(NTPDase), named NTPDase8, has been cloned and characterized. Analysis of cDNA reveals an openreading frame of 1491 base pairs encoding a protein of 497 amino acid residues with an estimated molecularmass of 54650 Da and a predicted isoelectric point of 5.94. In a mouse, the genomic sequence is locatedon chromosome 2A3 and is comprised of 10 exons. The deduced amino acid sequence reveals eightputative 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 NTPDase8hydrolyzed P2 receptor agonists, namely, ATP, ADP, UTP, and UDP, but did not hydrolyze AMP. Therewas an absolute requirement for divalent cations for the catalytic activity (Ca2+ > Mg2+) with an optimalpH between 5.5 and 8.0 for ATP and 6.4 for ADP hydrolysis. Kinetic parameters derived from analysisof crude cell lysates showed that the enzyme had lower apparent Km values for adenine nucleotides andfor triphosphonucleosides (Km,app of 13 
M for ATP, 41 M for ADP, 47 M for UTP, and 171 M forUDP). Hydrolysis of triphosphonucleosides resulted in a transient accumulation of the correspondingdiphosphonucleoside, as expected from the apparent Km values. Enzymatic properties of NTPDase8 differfrom those of other NTPDases suggesting an alternative way to modulate nucleotide levels and consequentlyP2 receptor activation.
M for ATP, 41 M for ADP, 47 M for UTP, and 171 M forUDP). Hydrolysis of triphosphonucleosides resulted in a transient accumulation of the correspondingdiphosphonucleoside, as expected from the apparent Km values. Enzymatic properties of NTPDase8 differfrom those of other NTPDases suggesting an alternative way to modulate nucleotide levels and consequentlyP2 receptor activation.