Localization of Na+/K(+)-ATPase in the bovine corneal endothelium.
A mouse monoclonal antibody has been used to localize Na+/K(+)-ATPase in the bovine corneal endothelium. The specificity of the antibody was demonstrated by reaction with a single protein of molecular mass 100 kDa on Western blots and immunoprecipitation of a complex consisting of 100 kDa and 50 kDa subunits. Treatment of the immunoprecipitated antigen with Peptide N-Glycanase F produced no change in the molecular mass of the 100 kDa protein, but resulted in a progressive decrease in the molecular mass of the 50 kDa subunit, to yield a core protein of molecular mass about 33 kDa. The pattern of deglycosylation suggested the presence of three N-linked glycans attached to the 33 kDa protein core. These results were consistent with the antibody being specific for the alpha subunit of the Na+/K(+)-ATPase. Immunocytochemical studies at the light and electron microscopic level demonstrated antibody binding to both the basal and lateral membranes of bovine corneal endothelial cells. This suggested a baso-lateral distribution of Na+/K(+)-ATPase in these cells, rather than the previously proposed lateral membrane-only distribution.
Guggenheim
J A
JA
Department of Optometry and Vision Sciences, University of Wales College of Cardiff, UK.
Hodson
S A
SA
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D003315:Cornea / Q000201:enzymology*; D004727:Endothelium / Q000201:enzymology; D006031:Glycosylation; D007150:Immunohistochemistry; D011233:Precipitin Tests; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis*
D000254:Sodium-Potassium-Exchanging ATPase