Functional expression of a cDNA encoding a human ecto-ATPase

1 The metabolism of extracellular nucleotides plays an important role in nu cleotide signalling mediated by P2 receptors. The nucleotide sequence encod ing a putative human ecto-ATPase named CD39L1 was reported recently. Howeve r, 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 CB3 9L1, with the exception that this sequence contains a 23 amino acid stretch in the putative extracellular loop that is missing in CD39L1. Partial sequ ence of a genomic DNA clone indicates that the CD39L1 gene corresponds to a n alternative spliced form of the human ecto-ATPase. 3 Stable expression of isolated sequence in NIH-3T3 mouse fibroblasts confe rred a marked nucleotide hydrolytic activity consistent with the activity o f an ecto-ATPase. 4 The human ecto-ATPase hydrolyzed all naturally occurring nucleoside triph osphates in a Ca2+- or Mg2+-dependent manner. Nucleoside diphosphates were hydrolyzed at a rate approximately 5% of that of the corresponding triphosp hates. The apparent K-m and V-max values were: 394+/-62 mu M and 107+/-7 nm ol Pi min(-1) 10(6) cells(-1) for the hydrolysis of ATP, and 102+/-33 mu M and 4+/-0.4 nmol Pi min(-1) 10(6) cells(-1) for the hydrolysis of ADP, resp ectively. 5 In conclusion, we report here the cloning and functional expression of a human ecto-ATPase. The study of the biochemical properties and the regulato ry mechanisms of ecto-ATPases of defined sequence will be valuable in the d efinition of their role in nucleotide signalling.