title: Purification, characterization, cloning, and expression of the chicken liver ecto-ATP-diphosphohydrolase.
abstract: 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 approximately 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.
authors: Department of Chemistry, San Diego State University, CA 92182-1030, USA. aknowles@chemistry.sdsu.edu
authors: Aileen F
authors: AF
authors: Knowles
authors: Agnes K
authors: AK
authors: Nagy
authors: Randy S
authors: RS
authors: Strobel
authors: Mae
authors: M
authors: Wu-Weis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D001081:Apyrase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data
chemical_list: D015703:Antigens, CD; D001081:Apyrase; C067869:CD39 antigen