The thermal unfolding and domain structure of Na+/K+-exchanging ATPase. A scanning calorimetry study.
The thermal unfolding and domain structure of Na+/K+-ATPase from pig kidney were studied by high-sensitivity differential scanning calorimetry (HS-DSC). The excess heat capacity function of Na+/K+-ATPase displays the unfolding of three cooperative domains with midpoint transition temperatures (Td) of 320.6, 327.5, 331.5 K, respectively. The domain with Td = 327.5 K was identified as corresponding to the beta subunit, while two other domains belong to the alpha subunit. The thermal unfolding of the low-temperature domain leads to large changes in the amplitude of the short-circuit current, but has no effect on the ATP hydrolysing activity. Furthermore, dithiothreitol or 2-mercaptoethanol treatment causes destruction of this domain, accompanied by significant disruption of the ion transporting function and a 25% loss of ATPase activity. The observed total unfolding enthalpy of the protein is rather low (approximately 12 J.g-1), suggesting that thermal denaturation of Na+/K+-ATPase does not lead to complete unfolding of the entire molecule. Presumably, transmembrane segments retain most of their secondary structure upon thermal denaturation. The binding of physiological ligands results in a pronounced increase in the conformational stability of both enzyme subunits.
Grinberg
A V
AV
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Vavilov St. 28, 117813 Moscow GSP-1, Russia.
Gevondyan
N M
NM
Grinberg
N V
NV
Grinberg
V Y
VY
D000818:Animals; D002152:Calorimetry, Differential Scanning; D007679:Kidney Medulla / Q000201:enzymology; D008024:Ligands; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013552:Swine; D013816:Thermodynamics
D008024:Ligands; D000254:Sodium-Potassium-Exchanging ATPase