Purification and immunohistochemical localization of the ATP diphosphohydrolase in bovine lungs.
We have recently described different isoforms of mammalian ATP diphosphohydrolase (ATPDase; EC 3.6.1.5). In the present study, we purified the lung ATPDase by column chromatographies followed by polyacrylamide gel electrophoresis under nondenaturing conditions. The active polypeptide that has a molecular mass of 78 kDa was identified by affinity labeling to the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA), followed by detection on Western blot with an antibody specific for FSBA. N-glycosidase F treatment shifted the molecular mass of the 78-kDa polypeptide down to 54 kDa, indicating that the enzyme bears approximately 6-12 NH2-linked oligosaccharide chains. A polyclonal antibody raised against the pancreas ATPDase, which specifically recognized the 78-kDa glycoprotein on Western blot, was used to carry out an immunological survey of the enzyme distribution in bovine lungs. Immunoreactivity was detected on airway epithelia from the trachea down to alveolar cells, airway and vascular smooth muscle cells, submucous glands, chondrocytes, leucocytes, as well as endothelial and mesothelial cells. Such a wide distribution suggests that the ATPDase may affect a variety of physiological effects mediated by extracellular nucleotides, such as airway smooth muscle tone, surfactant secretion, platelet aggregation, and inflammation.
Sévigny
J
J
Départment de biologie, Faculté des sciences, Université de Sherbrooke, Québec, Canada.
Picher
M
M
Grondin
G
G
Beaudoin
A R
AR
D000241:Adenosine / Q000031:analogs & derivatives; D000345:Affinity Labels; D000818:Animals; D001081:Apyrase / Q000145:classification / Q000302:isolation & purification* / Q000378:metabolism*; D002417:Cattle; D006023:Glycoproteins / Q000145:classification; D007150:Immunohistochemistry; D008168:Lung / Q000201:enzymology*; D008970:Molecular Weight; D014018:Tissue Distribution
D000345:Affinity Labels; D006023:Glycoproteins; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D001081:Apyrase; D000241:Adenosine