Isoform of Na+, K+-ATPase from rumen epithelium identified and quantified by immunochemical methods

Using biopsies of rumen epithelium papillae a net influx of [Rb-86(+)] was measured corresponding to a high concentration of Na+, K+-pumps found in [H-3]ouabain-binding studies (Kristensen el ai 1995). In the present study the Na+, K+-ATPase in papillae homogenates is compared with purified (Na+, K+)ATPase from different sources, immunochemically characterized with respect to the isoform of the hydrolytic a subunit and the concentration of pumps substantiated by a novel immunochemical method. Na+, K+-ATPase purified from bovine kidney was shown to contain one homogeneous high-affinity population of [H-3]ouabain-binding sites (K-d 1.37 nM). The ouabain-binding capacity was 0.82 nmol (mg protein)(-1). Site-directed polyclonal antibodies raised to isoform-specific sequences of the three known alpha-subunit isoforms and monoclonal alpha(1)-specific antibodies were used for isoform characterization on western blots of peptides separated by SDS-polyacrylamide gel electrophoresis. All three isoforms were present in Na+, K+-ATPase prepared from bovine brain. The alpha isoform of bovine kidney Na+, K+-ATPase and of rumen epithelium homogenate appeared to be alpha(l) whereas alpha(2) and alpha(3) were undetectable. Using an alpha(l)-specific antibody and (125)[-labelled antimouse IgG the content of (Na+, K+)-ATPase in rumen epithelium was determined by comparison of the signal from known amount of bovine kidney Na+, K+-ATPase on western blots. By this method rumen epithelium was found to contain 2.6 nmol Na+, K+-ATPase (g wet wt)(-1), i.e. a similarly high or even higher concentration than previously seen in ouabain-binding studies on biopsies.