Item

Abstract

In order to characterize the overall subunit interaction and the thermal stability of purified Na,K-ATPase isolated from pig kidney and dogfish (Squalus acanthias) rectal gland,1 a differential scanning calorimetry (DSC) study is carried out (Fig. 1a). With regard to ligand binding, the interaction stoichiometry (number of active sites n per protomer) and the affinity of ouabain and nucleotide binding are investigated by titration calorimetry at 25°C. Similar to our earlier results,2 the DSC thermogram of the pig kidney enzyme shows a single, very narrow, and almost symmetric endothermic denaturation transition at 57.0°C and a full width at half-maximum of 3.5°C. This is indicative of a uniform denaturation process of high cooperativity involving all subunits. Our result differs markedly from a recent investigation and thus questions the relevance of the interpretation postulated therein.3 The thermogram of the dogfish enzyme shows a lower transition temperature (49.5°C) and a much broader transition range than for the kidney enzyme. In addition, it exhibits a well-separated pretransition at 37.5°C. The pretransition is tentatively_assigned to the γ-like subunit of the dogfish enzyme. The broader transition can be an expression of less ordered subunit associations. The transition temperature of the micellar dogfish enzyme (C₁₂E₈ solubilized) is 8°C lower than that of the membrane-bound state. This can be due to an increased water access of the protein in the micellar state. All overall ΔH values are around 18 MJ/mol.