tailieunhanh - Báo cáo Y học: Purification, characterization, cloning, and expression of the chicken liver ecto-ATP-diphosphohydrolase

We previously demonstrated that the major ecto-nucleoside triphosphate phosphohydrolase in the chicken liver membranes is an ecto-ATP-diphosphohydrolase (ectoATPDase) [Caldwell, C., Davis, . & Knowles, . (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. . | Eur. J. Biochem. 269 2373-2382 2002 FEBS 2002 doi Purification characterization cloning and expression of the chicken liver ecto-ATP-diphosphohydrolase Aileen F. Knowles1 Agnes K. Nagy2 Randy S. Strobel3 and Mae Wu-Weis1 1 Department of Chemistry San Diego State University San Diego CA USA 2West Los Angeles Veterans Affairs Medical Center Los Angeles CA USA 3Department of Natural Sciences Metropolitan State University St Paul MN USA 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 . Knowles . 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 w 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 ectonucleoside triphosphate diphosphohydrolase E-NTPDase family. The stimulation of the expressed liver ecto-ATPDase by detergents indicates that