The thermal unfolding and domain structure of Na+/K+-exchanging ATPase - Ascanning calorimetry study

The thermal unfolding and domain structure of Na+/K+-ATPase from pig kidney were studied by high-sensitivity differential scanning calorimetry (HS-DSC ). The excess heat capacity function of Na+/K+-ATPase displays the unfoldin g of three cooperative domains with midpoint transition temperatures (T-d) of 320.6, 327.5, 331.5 K, respectively. The domain with T-d = 327.5 K was i dentified as corresponding to the beta subunit, while two other domains bel ong to the a subunit. The thermal unfolding of the lowtemperature domain le ads to large changes in the amplitude of the short-circuit current, but has no effect on the ATP hydrolysing activity. Furthermore, dithiothreitol. or 2-mercaptoethanol treatment causes destruction of this domain, accompanied by significant disruption of the ion transporting function and a 25% loss of ATPase activity. The observed total unfolding enthalpy of the protein is rather low (approximate to 12 J.g(-1)), suggesting that thermal denaturati on of Na+/K+-ATPase does not lead to complete unfolding of the entire molec ule. Presumably, transmembrane segments retain most of their secondary stru cture upon thermal denaturation. The binding of physiological ligands resul ts in a pronounced increase in the conformational stability of both enzyme subunits.