Addition of subunit gamma, K+ ions, and lipid restores the thermal stability of solubilized Na,K-ATPase

作者：Yoneda Juliana Sakamoto; Rigos Carolina Fortes; Ciancaglini Pietro^*

来源：Archives of Biochemistry and Biophysics, 2013, 530(2): 93-100.

DOI：10.1016/j.abb.2012.12.022

• Na,K-ATPase
• Solubilized protein
• Proteoliposome
• Thermal denaturation
• Calorimetry
• DSC

Differential scanning calorimetry (DSC) was applied to ascertain the effect caused by K+, Na+, ATP, detergent, DPPC, DPPE, and subunit gamma on the thermostability of Na,K-ATPase. The enthalpy variation (Delta H) for the thermal denaturation of the membrane-bound is twice the Delta H value obtained for solubilized Na,K-ATPase. Denaturation occurs in five steps for membrane-bound against three steps for the solubilized enzyme, therefore a multi-step unfolding process. In the presence of Na+, the melting temperature is 61.6 degrees C, and the Delta H is lower as compared with the Delta H obtained in the presence or in the absence of K+. Addition of ATP does not alter the transition temperatures significantly, but the shape of the curve is modified. Subunit gamma probably stabilizes Na,K-ATPase in the beginning of thermal unfolding, and different amounts of detergents in the solubilized sample change the protein stability. Reconstitution of Na,K-ATPase into a liposome shows that lipids exert a protector effect. These results reveal differences on the thermostability depending on the conformation of Na,K-ATPase. They are relevant because it allows a comparison with future studies, e.g. how the composition of the membrane interferes on the stability of Na,K-ATPase, elucidating the importance of the lipid type contained in cell membrane.

• 出版日期2013-2-15