Distribution, cloning, and characterization of porcine nucleoside triphosphate diphosphohydrolase-1.
In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung. The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions. Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non-covalent interactions.
Lemmens
R
R
Department of Medische Basiswetenschappen, Limburgs Universitair Centrum, Diepenbeek, Belgium.
Vanduffel
L
L
Kittel
A
A
Beaudoin
A R
AR
Benrezzak
O
O
Sévigny
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001081:Apyrase / Q000378:metabolism; D019556:COS Cells; D003001:Cloning, Molecular; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013552:Swine
D007527:Isoenzymes; D000251:Adenosine Triphosphatases; D011755:Pyrophosphatases; D001081:Apyrase; C021895:nucleoside triphosphate pyrophosphatase