Thermal denaturation of the Na,K-ATPase provides evidence for alpha-alpha oligomeric interaction and gamma subunit association with the C-terminal domain

Thermal denaturation can help elucidate protein domain substructure. We pre viously showed that the Na,K-ATPase partially unfolded when heated to 55 de greesC (Arystarkhova, E,, Gibbons, D. L,, and Sweadner, K, J. (1995) J, Bio l, Chem, 270, 8785-8796). The beta subunit unfolded without leaving the mem brane, but three transmembrane spans (M8-M10) and the C terminus of the alp ha subunit were extruded, while the rest of alpha retained its normal topol ogy with respect to the lipid bilayer, Here we investigated thermal denatur ation further, with several salient results. First, trypsin sensitivity at both surfaces of alpha was increased, but not sensitivity to V8 protease, s uggesting that the cytoplasmic domains and extruded domain were less tightl y packed but still retained secondary structure. Second, thermal denaturati on was accompanied by SDS-resistant aggregation of a subunits as dimers, tr imers, and tetramers without beta or gamma subunits, This implies specific alpha-alpha contact. Third, the gamma subunit, like the C-terminal spans of alpha, was selectively lost from the membrane. This suggests its associati on with M8-M10 rather than the more firmly anchored transmembrane spans. Th e picture that emerges is of a Na,K-ATPase complex of alpha, beta, and gamm a subunits in which a can associate in assemblies as large as tetramers via its cytoplasmic domain, while beta and gamma subunits associate with alpha primarily in its C-terminal portion, which has a unique structure and ther mal instability.