Functional expression of a cDNA encoding a human ecto‐ATPase

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. 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. Stable expression of isolated sequence in NIH‐3T3 mouse fibroblasts conferred a marked nucleotide hydrolytic activity consistent with the activity of an ecto‐ATPase. 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 K m and V max values were: 394±62 μ M and 107±7 nmol Pi min −1  10 6 cells −1 for the hydrolysis of ATP, and 102±33 μ M and 4±0.4 nmol Pi min −1  10 6 cells −1 for the hydrolysis of ADP, respectively. 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.British Journal of Pharmacology (1999) 128 , 396–402; doi: 10.1038/sj.bjp.0702805