Evidence for a diprotomeric structure of Na,K-ATPase. Accurate determination of protein concentration and quantitative end-group analysis.
Three methods were used to assess protein concentration in membrane-bound Na,K-ATPase preparations: standard Lowry assay, Kjeldahl nitrogen determination and amino acid analysis. While the first two methods showed excellent agreement, the third one always gave a lower value which varied drastically depending on the condition of sample treatment before amino acid analysis. This result reinforces the Lowry method in assessing the true concentration of Na,K-ATPase protein and suggests 250 kDa to be a true estimate of the molecular mass of the smallest ligand-binding unit of the enzyme. The cyanate method reveals two NH2-terminal residues of the beta-subunit (NH2-Ala) and one such residue of the alpha-subunit (NH2-Gly) per ligand-binding unit. From the data on equimolarity of the alpha- and beta-subunits in Na,K-ATPase this suggests that the enzyme molecule is composed of two alpha beta-protomers, one possessing a modified (presumably an N-blocked) alpha-subunit.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D007679:Kidney Medulla / Q000201:enzymology; D008722:Methods; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis*; D013552:Swine
D000596:Amino Acids; D000254:Sodium-Potassium-Exchanging ATPase