Molecular Cloning of the Chicken Oviduct Ecto-ATP-Diphosphohydrolase

Abstract

The chicken oviduct ecto-ATP diphosphohydrolase (ATPDase), a member of the ecto-ATPase family, was purified to homogeneity previously (Strobel, R. S., Nagy, A. K., Knowles, A. F., Buegel, J., and Rosenberg, M. O. (1996) J. Biol. Chem. 271, 16323-16331). It is an 80-kDa glycoprotein with high specific activity (approximately 1,000 mol/min/mg with MgATP as the substrate) and hydrolyzes both nucleoside triphosphates and diphosphates. Using amino acid sequence information obtained from the purified enzyme, two partial cDNA clones were obtained using reverse transcriptase-polymerase chain reaction and library screening. This is the second ecto-ATPase family member and the first ecto-ATPDase to be cloned from information derived from purified proteins. The deduced primary sequence of the chicken oviduct ecto-ATPDase indicates a protein of 493 amino acid residues with a molecular mass of 54 kDa. The predicted orientation shows it to be anchored to the membrane by two transmembranous segments near the NH 2 and COOH termini with very short intracytoplasmic peptides at either end. The bulk of the protein is extracellular and contains 12 potential N-glycosylation sites, several potential phosphorylation sites, and five sequences that are conserved in seven other related membrane proteins. Four of the conserved sequences, designated as apyrase conserved regions, are present in both ecto-ATPases and soluble E-type ATPases. The fifth conserved region, which occurs near the COOH terminus of the eight proteins, is observed only in the membrane-bound ecto-ATPases. Unexpectedly, sequence comparison revealed that the chicken oviduct ecto-ATPDase is equally distant from the two ecto-ATPases, which exhibit low activity toward ADP, and the four putative ecto-ATPDases, which are closely related to CD39.

Purification, characterization, cloning, and expression of the chicken liver ecto‐ATP‐diphosphohydrolase

“…This proved to be the case when we obtained the cDNA of the chicken liver ecto-ATPDase by RT-PCR cloning. The two chicken ecto-ATPDases differ in only seven amino acids out of 493 amino-acid residues, all located in the C-terminal half of the molecules that are less conserved in the E-ATPases [21]. There is an additional N-glycosylation site in the liver ecto-ATPDase.…”

“…55 kDa [21]. It seems likely that the two enzymes may have similar primary sequences despite the different molecular masses of the native enzymes.…”

“…5) is nearly identical to that of the oviduct ectoATPDase differing in seven amino acids out of 493 amino acids. It has the same two transmembranous domains, five apyrase conserved regions (ACRs), 10 conserved cysteine residues, and 12 potential N-glycosylation sites as the oviduct ecto-ATPDase [21]. Northern blot analysis using total chicken liver RNA revealed a major transcript of 2 kb (data not shown).…”

“…We previously reported the immunoaffinity purification of the chicken oviduct ecto-ATPDase to homogeneity [7] and its molecular cloning [21]. An ecto-ATPDase similar to the chicken oviduct enzyme is present in the chicken liver membranes [23].…”

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

“…Purinergic receptors: Select P2X and P2Y purinergic receptors are expressed by hepatocytes (Figure 1) Human and rat NTPDase8 cDNA has been cloned and the genes are located on chromosome loci 9q34 and 3p13, respectively (71,89). NTPDase8 resembles the chicken ecto-ATPase cloned from oviduct and liver (90,91). Major ATPase and ADPase activity was detected in the bile canaliculi by immunohistochemistry after incubation with nucleotides.…”