Identification, characterization, and immunolocalization of a nucleoside triphosphate diphosphohydrolase in pig liver

Different isoforms of nucleoside triphosphate diphosphohydrolases (NTPDases ; EC 3.6.1.5), also identified as ATP diphosphohydrolases, have been previo usly described in mammalian tissues. We report here the biochemical charact erization of NTPDases in the pig liver. Optimum pH of catalysis is more aci dic for this enzyme than for NTPDases (neutral or alkaline pH) found in oth er mammalian tissues. It is less sensitive to bile salts than the bovine sp leen NTPDase. Calculated K-m values for ATP and ADP (31 and 21 mu M, respec tively) are slightly higher than those reported for the latter enzyme. Elec trophoretograms of these enzymes also show different migration patterns. We stern blots with Ringo, an antibody that recognizes the different isoforms of mammalian NTPDases, show a small but reproducible difference in estimate d molecular masses (75 kDa for liver vs 78 kDa for spleen NTPDase). A secon d antibody, generated against a different sequence of NTPDase I, does not r ecognize the liver enzyme, thereby indicating some differences in primary s tructure. Immunolocalization produced a strong signal on hepatocytes, epith elial cells of the bile duct system, and vascular cells. Immunoreactivity w as variable among hepatocytes of different lobules and among hepatocytes wi thin a given lobule. In general, those located in the perilobular zone were more reactive than those located in the central zone and in the periphery of the centrolobular vein. (C) 2000 Academic Press.