The thermal unfolding and domain structure of Na+/K+âexchanging ATPase.â: A scanning calorimetry study
2001
The thermal unfolding and domain structure of Na+/K+âATPase from pig kidney were studied by highâsensitivity differential scanning calorimetry (HSâDSC). The excess heat capacity function of Na+/K+âATPase displays the unfolding of three cooperative domains with midpoint transition temperatures (Td) of 320.6, 327.5, 331.5 K, respectively. The domain with Tdâ=â327.5 K was identified as corresponding to the β subunit, while two other domains belong to the α subunit. The thermal unfolding of the lowâtemperature domain leads to large changes in the amplitude of the shortâcircuit current, but has no effect on the ATP hydrolysing activity. Furthermore, dithiothreitol or 2âmercaptoethanol 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 (≈â12âJ·g−1), suggesting that thermal denaturation of Na+/K+âATPase does not lead to complete unfolding of the entire molecule. Presumably, transmembrane segments retain most of their secondary structure upon thermal denaturation. The binding of physiological ligands results in a pronounced increase in the conformational stability of both enzyme subunits.
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