An ecto-ATP diphosphohydrolase (ATPDase) was purified to homogeneity from vesiculosomes shed from chicken oviduct. First, the ecto-ATPDase-enriched vesiculosomes were concentrated by filtration, differential centrifugation, and exclusion chromatography. Next, the nonionic detergent, Nonidet P-40, was used to extract the ecto-ATPDase from vesiculosomal membranes, and the solubilized enzyme was further purified by ion exchange (DEAE-Bio-Gel) and lentil-lectin-Sepharose 4B chromatography. In the final stage, immunoaffinity chromatography was utilized to obtain purified ecto-ATPDase. More than 25,000-fold purification was achieved. Specific activity of the purified enzyme was greater than 800 mol/min/mg of protein with MgATP as the substrate, the highest ever reported for an ATPDase. The enzyme also hydrolyzed other nucleoside triphosphates in the presence of magnesium at similar rates and CaATP and MgADP at lower rates. The molecular mass of the purified glycoprotein was 80 kDa as determined by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Based on its enzymatic properties, the relationship of the chicken oviduct ecto-ATPDase with other reported ATPDases and ecto-ATPases is discussed.The chicken oviduct has been used as a model system for studying secretion (1). Enzymes which are postulated to be involved in the egg formation process, such as an ecto-protein kinase (2), various phosphatases (3), and an ATPase have been investigated by Rosenberg et al. (4). The ATPase, first described by Haaland and Rosenberg (5), showed extensive kinetic similarities to the Mg 2ϩ

The chicken gizzard smooth muscle extracellular ATPase (ecto-ATPase) is a low abundance, high specific activity, divalent cation-dependent, nonspecific nucleotide triphosphatase (NTPase). The ATPase is a 66-kDa glycoprotein with a protein core of 53 kDa (Stout, J.G. and Kirley, T.L. (1994) J. Biochem. Biophys. Methods 29, 61-75). In this study we evaluated the characteristics of a bank of monoclonal antibodies raised against a partially purified chicken gizzard ecto-ATPase. 18 monoclonal antibodies identified by an ATPase capture assay were tested for effects on ATPase activity as well as for their Western blot and immunoprecipitation potential. The five most promising monoclonal antibodies were used to immunopurify the ecto-ATPase. The one-step immunoaffinity purification of solubilized chicken gizzard membranes with all five of these monoclonal antibodies isolated a 66-kDa protein whose identity was confirmed by N-terminal sequence analysis to be the ecto-ATPase. Several of these monoclonal antibodies stimulated ecto-ATPase activity similar to that observed previously with lectins. Western blot analysis revealed that three of the five monoclonal antibodies recognized a major immunoreactive band at 66 kDa (53-kDa core protein), consistent with previous purification results. The other two antibodies recognized proteins of approximately 90 and 160 kDa on Western blots. The 90-kDa co-immunopurifying (and presumably associated or related) protein was identified by N-terminal analysis as LEP100, a glycoprotein that shuttles between the plasma and lysosomal membranes. The approximately 160-kDa co-immunopurifying protein was identified by N-terminal analysis as integrin, a protein involved in extracellular contacts with adhesion molecules. Extended N-terminal sequence analysis of the immunopurified 66-kDa ecto-ATPase revealed some sequence homology with mouse lysosomal associated membrane protein. Tissue distribution of the ecto-ATPase showed that the highest levels of protein were expressed in muscle tissues (cardiac, skeletal, and smooth) and brain.

We previously demonstrated that the major ecto‐nucleoside triphosphate phosphohydrolase in the chicken liver membranes is an ecto‐ATP‐diphosphohydrolase (ecto‐ ATPDase) [Caldwell, C., Davis, M.D. & Knowles, A.F. (1999) Arch. Biochem. Biophys. 362, 46–58]. Enzymatic properties of the liver membrane ecto‐ATPDase are similar to those of the chicken oviduct ecto‐ATPDase that we have previously purified and cloned. Using antibody developed against the latter, we have purified the chicken liver ecto‐ATPDase to homogeneity. The purified enzyme is a glycoprotein with a molecular mass of 85 kDa and a specific activity of ≈ 1000 U·mg protein−1. Although slightly larger than the 80‐kDa oviduct enzyme, the two ecto‐ATPDases are nearly identical with respect to their enzymatic properties and mass of the deglycosylated proteins. The primary sequence of the liver ecto‐ATPDase deduced from its cDNA obtained by RT‐PCR cloning also shows only minor differences from that of the oviduct ecto‐ATPDase. Immunochemical staining demonstrates the distribution of the ecto‐ATPDase in the bile canaliculi of the chicken liver. HeLa cells transfected with the chicken liver ecto‐ATPDase cDNA express an ecto‐nucleotidase activity with characteristics similar to the enzyme in its native membranes, most significant of these is stimulation of the ATPDase activity by detergents, which inhibits other members of the ecto‐ nucleoside triphosphate diphosphohydrolase (E‐NTPDase) family. The stimulation of the expressed liver ecto‐ATPDase by detergents indicates that this property is intrinsic to the enzyme protein, and cannot be attributed to the lipid environment of the native membranes. The molecular identification and expression of a liver ecto‐ATPDase, reported here for the first time, will facilitate future investigations into the differences between structure and function of the different E‐NTPDases, existence of liver ecto‐ATPDase isoforms in different species, its alteration in pathogenic conditions, and its physiological function.

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