Heterogeneity of Na,K-ATPase from kidney.
Na,K-ATPase was purified from pig and mink kidney. Enzyme batches of high specific activity apparently contained only alpha- and beta-peptides, judging from the SDS-gel-electrophoretic pattern. Careful determinations of the ouabain-binding capacity and binding kinetics were carried out. The question whether the functional unit is an alpha beta-protomer, a diprotomer or even a polymer was analysed from the binding stoichiometry and the binding pattern at nonsaturating ouabain concentrations. The binding capacity exceeded half of the theoretical maximum value for exclusively alpha beta-promoters. A half-of-the-sites reactivity of a diprotomer thus seems less likely, though nearly half of the protein is not accounted for in its absence. When ouabain binding took place in the presence of Na+, at least two binding components were noticed. Most probably the two components are not of equal size, thus excluding a co-operative diprotomer construction of the active ouabain-binding units. If the co-operativity model has to be abandoned, the phenomenon could be due to two or more isozymes exhibiting different Na(+)-affinities. Isoforms of the hydrolytic alpha-peptide were analysed by an ELISA technique and on blots of Na,K-ATPase peptides utilizing commercial isoform-specific antibodies. Though not absolutely specific, the individual antibodies were assumed to be monospecific with respect to the single isoforms of the alpha-peptide. In that case, data were consistent with a significant contribution of alpha 2- and alpha 3-isoforms (about 15%), in addition to the predominant alpha 1-isoform in kidney Na,K-ATPase.
Hansen
O
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Biomembrane Research Centre, Aarhus University, Denmark.
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007527:Isoenzymes / Q000737:chemistry*; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D008907:Mink / Q000378:metabolism*; D010042:Ouabain / Q000378:metabolism; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013552:Swine
D007527:Isoenzymes; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase