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  • Title: Sulphydryl groups of (Na+ + K+)-ATPase from rectal glands of Squalus acanthias. Detection of ligand-induced conformational changes.
    Author: Esmann M.
    Journal: Biochim Biophys Acta; 1982 May 21; 688(1):260-70. PubMed ID: 6284233.
    Abstract:
    1. Modification of the Class II sulphydryl groups on the (Na+ + K+)-ATPase from rectal glands of Squalus acanthias with N-ethylmaleimide has been used to detect conformational changes in the protein. The rates of inactivation of the enzyme and the incorporation of N-ethylmaleimide depend on the ligands present in the incubation medium. With 150 mM K+ the rate of inactivation is largest (k1 = 1.73 mM-1 . min-1) and four SH groups per alpha-subunit are modified. The rate of inactivation in the presence of 150 mM Na+ is smaller (k1 = 1.08 mM-1 . min-1) but the incorporation of N-ethylmaleimide is the same as with K+. 2. ATP in micromolar concentrations protects the Class II groups in the presence of Na+ (k1 = 0.08 mM-1 . min-1 at saturating ATP) and the incorporation is drastically reduced. ATP in millimolar concentrations protects the Class II groups partially in the presence of K+ (k1 = 1.08 mM-1 . min-1) and three SH groups are labelled per alpha subunit. 3. The K+-dependent phosphatase is inhibited in parallel to the (Na+ + K+)-ATPase under all conditions, and the ligand-dependent incorporation of N-ethylmaleimide was on the alpha-subunit only. 4. It is shown that the difference between the Na+ and K+ conformations sensed with N-ethylmaleimide depends on the pH of the incubation medium. At pH 6 there is a very small difference between the rates of inactivation in the presence of Na+ and K+, but at higher pH the difference increases. It is also shown that the rate of inactivation has a minimum at pH 6.9, which suggests that the conformation of the enzyme changes with pH. 5. Modification of the Class III groups with N-ethylmaleimide--whereby the enzyme activity is reduced from about 16% to zero--shows that these groups are also sensitive to conformational changes. As with the Class II groups, ATP in micromolar concentrations protects in the presence of Na+ relative to Na+ or K+ alone. ATP in millimolar concentrations with K+ present increases the rate of inactivation relative to K+ alone, in contrast to the effect on the Class II groups. 6. Modification of the Class II groups with a maleimide spin label shows a difference between Class II groups labelled in the presence of Na+ (or K+) and Class II groups labelled in the presence of K + ATP, in agreement with the difference in incorporation of N-ethylmaleimide. The spectra suggest that the SH group protected by ATP in the presence of K+ is buried in the protein. 7. The results suggest that at least four different conformations of the (Na+ + K+)-ATPase can be sensed with N-ethylmaleimide: (i) a Na+ form of the enzyme with ATP bound to a high-affinity site (E1-Na-ATP); (ii) a Na+ form without ATP bound (E1-Na); (iii) a K+ form without ATP bound (E2-K); and (iv) an enzyme form with ATP bound to a low-affinity site in the presence of K+, probably and E1-K-ATP form.
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