IMMUNOLOCALIZATION OF THE ECTO-ATPASE AND ECTO-APYRASE IN CHICKEN GIZZARD AND STOMACH - PURIFICATION AND N-TERMINAL SEQUENCE OF THE STOMACHECTO-APYRASE

We have examined the in vivo localization of extracellular ecto-ATPase and ecto-apyrase (ATPDase) in adult chicken gizzard and stomach by im munofluorescence and laser scanning confocal microscopy. In chicken gi zzard, the ecto-ATPase was distributed in discrete clusters restricted to the sarcolemma of the smooth muscle cells, Anti-ecto-apyrase antib ody detected a single 80-kDa band (putative apyrase) in Western blots of both chicken gizzard membrane extracts and partially purified anion exchange fractions, but the antibody did not detect ecto-apyrase in i mmunolabeled gizzard cryosections. In adult chicken stomach, the ecto- apyrase was observed at the apical membrane of the glandular oxyntico- peptic cells as described in previous immunoperoxidase studies (Stout, J. G., R. S. Strobel, and T. L. Kirley (1995) Biochem, Mol. Biol, Int . 36, 529-535). However, ecto-ATPase was clustered in the sarcolemma o f the organized layer of circular smooth muscle and in smooth muscle c ells of the septa surrounding the glandular tissue, but not in the gla ndular cells containing the ecto-apyrase, The findings indicate compar tmentalization of the two related extracellular nucleotide hydrolyzing enzymes and suggest differential functions that are specialized for d ifferent regions of the chicken stomach, We also partially purified th e ecto apyrase of chicken stomach, an 80 kDa membrane glycoprotein. Ch icken stomach membranes were solubilized in digitonin, glycoproteins w ere separated from solubilized proteins by lectin chromatography, and nucleotide-binding glycoproteins were selected by immobilized Cibacron blue chromatography, Further purification by size exclusion and anion exchange chromatography yielded purification of 94-fold, The ATPase s pecific activity of the purified stomach ecto-apyrase was 75,000 mu mo l of P-i/mg of protein/h, and the purified preparation consisted of a major band (55% of total protein) at 80 kDa, The purified enzyme could be deglycosylated with peptide N-glycosidase-F to a core molecular ma ss of 54 kDa. The N-terminal sequence of the 80-kDa stomach ecto-apyra se band (which reacted with anti-ecto-ATPDase antibodies) was determin ed to be: MEYKGKVVAGLLTATWV. Immunological cross-reactivity data indic ate that the stomach 80-kDa protein isolated is an ecto apyrase and is related to both the chicken liver and oviduct ecto-ATPDase enzymes ch aracterized earlier, as well as to the human lymphoid cell activation antigen, CD39.