Immunolocalization of the Ecto-ATPase and Ecto-apyrase in Chicken Gizzard and Stomach

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 ( 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 mol 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 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.

Molecular Cloning of the Chicken Oviduct Ecto-ATP-Diphosphohydrolase

“…It differs from the ecto-ATPDases in that it does not hydrolyze ADP. Immunohistochemical studies further showed that although both ecto-ATPase and ecto-ATPDase are present in the chicken stomach, they have distinctly different localization, implying possibly different functions (15). Nevertheless, the deduced amino acid sequence of the chicken gizzard ecto-ATPase revealed significant sequence homology to that of CD39.…”

“…An aliquot of the cDNA was amplified by polymerase chain reaction (PCR) using AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA) with 1 M primers and 2 mM MgCl 2 . Degenerate oligonucleotides, 5Ј-ATGGAR-TAYAARGGNAARGT-3Ј (sense, NH1, nucleotides [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] corresponding to the amino acid sequence of the NH 2 -terminal peptide, MEYKGKV, and 5Ј-RAARTCNACNGGRAAYTC-3Ј (antisense, NH4, nucleotides 465-448) corresponding to the amino acid sequence of the internal peptide, EFPVDF, were used as primers. The reaction mixture was heated for 2 min at 95°C before addition of the Taq polymerase, followed by thermal cycling at 95°C for 45 s, 47°C for 30 s, and 70°C for 3 min for 4 cycles; then 95°C for 45 s, 53°C for 30 s, 70°C for 3 min for 30 cycles; and last, 1 cycle at 95°C for 30 s, 53°C for 30 s and 70°C for 10 min.…”

“…Ecto-ATPases from rabbit transverse tubules (6), human umbilical vessel (7), chicken oviduct (8,9), chicken liver (8), chicken gizzard (10), and human placenta (11) have been purified to homogeneity. Extensive purification of ecto-ATPases from pig pancreas (12), bovine lung (13), bovine aorta (14), and chicken stomach (15) have also been reported.…”

The Transmembrane Domains of Ectoapyrase (CD39) Affect Its Enzymatic Activity and Quaternary Structure

“…of P i /min/mg) is lower than that of the digitonin-solubilized rat lung ectoapyrase (ϳ1000 mol of P i /min/mg of protein) (39).…”

“…Second, sucrose density gradient sedimentation profiles of both the expressed ectoapyrase in COS-7 cells and the native ectoapyrases from LG2 cells (data not shown) are identical: ectoapyrase behaved as a tetramer after solubilization with digitonin, a detergent that has little effect on the enzymatic activity, and became monomeric after solubilization with Triton X-100, a detergent that inhibits the activity. It has been suggested that detergents may inactivate ectoapyrase by destabilizing an oligomeric state of the protein (33,34), and it has been reported that chicken gizzard muscle ecto-ATPase (34) and rat lung ectoapyrase (39) are disulfide-linked homotrimers and homodimers, respectively. However, this work is the first demonstration that the ectoapyrase is a noncovalent tetramer.…”