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.
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