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

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Abstract
Using biopsies of rumen epithelium papillae a net influx of [86Rb+] was measured corresponding to a high concentration of Na+, K+-pumps found in [3H]ouabain-binding studies ( Kristensen et al. 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 α 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 [3H]ouabain-binding sites (Kd 1.37 n M). The ouabain-binding capacity was 0.82 nmol (mg protein)−1. Site-directed polyclonal antibodies raised to isoform-specific sequences of the three known α-subunit isoforms and monoclonal α1-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 α isoform of bovine kidney Na+, K+-ATPase and of rumen epithelium homogenate appeared to be α1 whereas α2 and α3 were undetectable. Using an α1-specific antibody and 125I-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.