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Study of Structure‐Functional Organization Features in the Pig Kidney Na,K‐ATPase at Different Conformational States

The Na,K-ATPase reaction cycle is a chain of consecutive correlated conformational transitions in the enzyme molecule induced by Na-dependent phosphorylation and K-dependent dephosphorylation conjugated with occlusion and translocation of sodium and potassium ions through the plasma membrane. Every stage in this process is characterized by a unique, thermodynamically stable structure of polypeptide chain organization; the description of this organizational peculiarity may be key to interpreting enzyme function. Despite extensive studies of enzyme function at various stages, little is known about the enzyme’s thermodynamic potential or the molecular mechanisms underlying its cyclic conformational transitions. This work is dedicated to the study of those aspects of Na,K-ATPase function. For this purpose we chose two approaches: measurement of thermodynamic characteristics of the various enzyme structural domains and analysis of reactivity of SH groups affected by intramolecular ligand-dependent conformational transitions. The thermal unfolding and domain structure of the Na,K-ATPase from pig kidney were studied by high-sensitivity differential scanning calorimetry. We demonstrated that thermal denaturation of Na,K-ATPase was displayed in the unfolding of three independent cooperative units (domains 1,2, and 3) with midpoint transition temperatures (Td) of 320.6,327.5, and 331.5 K, respectively. Domains 1 and 3 were identified as corresponding to the (Y subunit, domain 2 to the p subunit. The total enthalpy of ion-free native Na,K-ATPase is 1,753 kT/mol or 2.8 caYg, including ly subunit enthalpy 1,259 kTtmol(340 kTlmol for domain 1 and 919 Idlmol for domain 3) and p subunit enthalpy 494 kJ/mol. To clarify whether conformations of the domains are affected by natural ligands, we examined the thermal unfolding of membrane-bound Na,K-ATPase with monovalent cations Na+ and K+ and with ATP. Na+ ions induce a conformational transition, stabilizing the enzyme structure, with total enthalpy increase of 155 kJ/mol. The main increase (105 kJ/mol) corresponds to domain 1 of (Y subunit. Domains 2 and 3 are