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.