Urea-induced unfolding of Na,K-ATPase as evaluated by electron paramagnetic resonance spectroscopy.
Urea-induced unfolding of Na,K-ATPase from pig kidney and from shark salt gland was studied by electron paramagnetic resonance (EPR) spectroscopy of a nitroxyl derivative of maleimide covalently attached to sulfhydryl groups which are essential for activity. Urea-induced structural changes lead to the inhibition of Na,K-ATPase activity. Structural changes detected by EPR are reversible over the whole range of urea concentrations (0-8 M), although activity loss is always irreversible. The structure of the cytoplasmic domain is more accessible and more susceptible to perturbations than is the transmembrane sector of the Na,K-ATPase and thus is more sensitive to denaturant. Conformational changes at the active thiol groups of these enzymes indeed take place before unfolding of the enzyme as a whole, together with enzyme inactivation. Na,K-ATPase from pig kidney is more stable not only to thermal denaturation but also to urea-induced denaturation than is the Na,K-ATPase from shark salt gland. Susceptibility of the latter could arise from the nonhomologous regions in the cytoplasmic domain.
Babavali
Mohammad
M
Department of Physiology and Biophysics, Aarhus University, Aarhus, Denmark.
Esmann
Mikael
M
Fedosova
Natalya U
NU
Marsh
Derek
D
D000818:Animals; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D008958:Models, Molecular; D011489:Protein Denaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012491:Salt Gland / Q000201:enzymology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D013113:Spin Labels; D048251:Squalus acanthias; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013552:Swine; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D004791:Enzyme Inhibitors; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D014508:Urea; D000254:Sodium-Potassium-Exchanging ATPase