Abstract
We have examined the in vivo localization of extracellular ecto-ATPase and ecto-apyrase (ATPDase) in adult chicken gizzard and stomach by immunofluorescence and laser scanning confocal microscopy. In chicken gizzard, the ecto-ATPase was distributed in discrete clusters restricted to the sarcolemma of the smooth muscle cells. Anti-ecto-apyrase antibody 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 immunolabeled 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 of the organized layer of circular smooth muscle and in smooth muscle cells of the septa surrounding the glandular tissue, but not in the glandular cells containing the ecto-apyrase. The findings indicate compartmentalization of the two related extracellular nucleotide hydrolyzing enzymes and suggest differential functions that are specialized for different regions of the chicken stomach. We also partially purified the ecto-apyrase of chicken stomach, an 80-kDa membrane glycoprotein. Chicken stomach membranes were solubilized in digitonin, glycoproteins were 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 specific activity of the purified stomach ecto-apyrase was 75,000 micromol of Pi/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 mass of 54 kDa. The N-terminal sequence of the 80-kDa stomach ecto-apyrase band (which reacted with anti-ecto-ATPDase antibodies) was determined to be: MEYKGKVVAGLLTATWV. Immunological cross-reactivity data indicate that the stomach 80-kDa protein isolated is an ecto-apyrase and is related to both the chicken liver and oviduct ecto-ATPDase enzymes characterized earlier, as well as to the human lymphoid cell activation antigen, CD39.
Highlights
We have examined the in vivo localization of extracellular ecto-ATPase and ecto-apyrase (ATPDase) in adult chicken gizzard and stomach by immunofluorescence and laser scanning confocal microscopy
The findings indicate compartmentalization of the two related extracellular nucleotide hydrolyzing enzymes and suggest differential functions that are specialized for different regions of the chicken stomach
Chicken stomach membranes were solubilized in digitonin, glycoproteins were separated from solubilized proteins by lectin chromatography, and nucleotide-binding glycoproteins were selected by immobilized Cibacron blue chromatography
Summary
Materials—Tissue from adult chickens (female Ross Avian 24) was obtained fresh from a local poultry slaughterhouse. 5 mg of adult chicken gizzard or stomach membrane protein was solubilized at 1.0 mg/ml with 1% digitonin in 20 mM MOPS and 2 mM MgCl2, pH 7.4. The solubilized proteins were loaded unto a QMA MemSep membrane anion exchanger (size 1000) equilibrated in 0.1% digitonin in 10 mM Tris and 2 mM MgCl2, pH 8.2 (buffer B), at 2 ml/min. The solution was gently mixed at room temperature for 15 min and centrifuged at 48,000 rpm in a 50Ti rotor for 45 min, and the supernatant was diluted to 0.1% digitonin with the MOPS/MgCl2 buffer. The PVDF membranes were blocked for 1 h in 5% milk and 0.02% NaN3 in Tris-buffered saline and incubated overnight at room temperature in monoclonal antibodies mAb 15 (anti-chicken gizzard ecto-ATPase antibody; Ref. 18), or MC18 (anti-chicken oviduct ecto-ATPDase (apyrase); Ref. 20). The slides were cover-slipped in a diazabicyclo(2.2.2)octane 70% glycerol mounting media and examined using a Bio-Rad MRC 600 laser scanning confocal microscope and CoMOS image analysis software
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