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

AbstractHeat 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 αβ-protomers (Mr≈140000). 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 ≈ 230 kJmol higher. The data suggest that (i) in a functionally active enzyme form, the αβ-protomers possess a rigid structure with tight association of their subunits and domains, (ii) this structural rigidity is essential for the Na+,K+-ATPase functioning and (iii) there is a unique non-active conformation of the enzyme which may play an important role in its in vivo regulation