Cooperativity of the alpha beta-protomer structure in Na+,K+-ATPase functioning. A scanning microcalorimetry study

Heat denaturation of the free and ligand-bound forms of purified Na+,K+-ATPase from pig kidney is studied with the scanning microcalorimetry technique. A single two-state transition is observed during denaturation of the free enzyme, the molar concentration of the cooperatively melting units being equal to the concentration of alpha beta-protomers (Mr approximately equal to 140 000). Upon interaction of the enzyme with phosphate, Mg2+, and strophanthidin, but not with Na+, the cooperativity of the protomer unfolding is lost, and the protein stabilization enthalpy becomes approximately equal to 230 kJ/mol higher. The data suggest that in a functionally active enzyme form, the alpha beta-protomers possess a rigid structure with tight association of their subunits and domains, this structural rigidity is essential for the Na+,K+-ATPase functioning and there is a unique non-active conformation of the enzyme which may play an important role in its in vivo regulation.