Functional expression of a cDNA encoding a human ecto-ATPase.
1 The metabolism of extracellular nucleotides plays an important role in nucleotide signalling mediated by P2 receptors. The nucleotide sequence encoding a putative human ecto-ATPase named CD39L1 was reported recently. However, the biological activity of this protein has not been established. 2 Based on the sequence of CD39L1 we isolated from mRNA from human ECV-304 cells a sequence encoding a 495 amino acid protein that is identical to CD39L1, with the exception that this sequence contains a 23 amino acid stretch in the putative extracellular loop that is missing in CD39L1. Partial sequence of a genomic DNA clone indicates that the CD39L1 gene corresponds to an alternative spliced form of the human ecto-ATPase. 3 Stable expression of isolated sequence in NIH-3T3 mouse fibroblasts conferred a marked nucleotide hydrolytic activity consistent with the activity of an ecto-ATPase. 4 The human ecto-ATPase hydrolyzed all naturally occurring nucleoside triphosphates in a Ca(2+)- or Mg(2+)-dependent manner. Nucleoside diphosphates were hydrolyzed at a rate approximately 5% of that of the corresponding triphosphates. The apparent Km and Vmax values were: 394+/-62 microM and 107+/-7 nmol Pi min-1 10(6) cells-1 for the hydrolysis of ATP, and 102+/-33 microM and 4+/-0.4 nmol Pi min-1 10(6) cells-1 for the hydrolysis of ADP, respectively. 5 In conclusion, we report here the cloning and functional expression of a human ecto-ATPase. The study of the biochemical properties and the regulatory mechanisms of ecto-ATPases of defined sequence will be valuable in the definition of their role in nucleotide signalling.
Mateo
J
J
Department of Pharmacology, The School of Medicine, University of North Carolina, Chapel Hill, North Carolina, NC 27599, USA.
Harden
T K
TK
Boyer
J L
JL
D000251:Adenosine Triphosphatases / Q000096:biosynthesis* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010710:Phosphates / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D010710:Phosphates; D000251:Adenosine Triphosphatases; C027201:ectoATPase