Isoform of Na+, K(+)-ATPase from rumen epithelium identified and quantified by immunochemical methods.
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 alpha 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 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 1 whereas alpha 2 and alpha 3 were undetectable. Using an alpha 1-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.
Hansen
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Department of Physiology, Aarhus University, Denmark.
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D001923:Brain Chemistry; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000737:chemistry; D006868:Hydrolysis; D007120:Immunochemistry / Q000379:methods*; D007527:Isoenzymes / Q000302:isolation & purification*; D007668:Kidney / Q000737:chemistry; D012417:Rumen / Q000737:chemistry*; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification*
D007527:Isoenzymes; D000254:Sodium-Potassium-Exchanging ATPase