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10th International Congress "Cell Volume Regulation: Novel Therapeutic Targets and Pharmacological Approaches"
DIFFERENT CONFORMATIONS OF NA,K-ATPASE INDUCED BY THE BINDING OF OUABAIN AND MARINOBUFAGENIN
Klimanova, E.A.1, Petrushanko, I.Yu.2, Mitkevich, V.A.2, Akimova, O.A.1, Orlov, S.N.1, and Lopina, O.D.1
1 Department of Biochemistry, School of Biology, M.V. Lomonosov Moscow State University, Moscow, Russian Federation
2 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation
Na,K-ATPase plays an important role in the regulation of cell volume affecting the activity of different ion-exchangers. Numerous data demonstrate that this enzyme is also receptor for cardiotonic steroids (CTS) that without pump inhibition can activate different signal cascades. CTS ouabain and marinobufagenin are known to inhibit Rb+ transport providing by Na,K-ATPase in kidney epithelial cells (MDCK) with similar values of IC50 equal to about 0,1 |M [1]. Simultaneously ouabain and marinobufagenin induce cell death on the way characterized by mixed sign of necrosis (cell swelling) and apopto-sis (activation of caspase-3) with different values of IC50 (about 0.05 and 5 |M respectively). The data suggest that different CTS may induce different conformation of Na,K-ATPase that, in turn, may provides the binding to the enzyme different proteins activating signal cascades. Using purified Na,K-ATPase from rabbit kidney we have found that ouabain and marinobufagenin inhibited Na,K-ATPase by similar manner. However, it was shown using Na,K-ATPase labeled with 5-iodoacetamidofluorescein [2] that the conformations of Na,K-ATPase induced by
ouabain and marinobufagenin are different. Using isothermal titration calorimetry (ITC) we have found that at 25 °C the binding constants for marinobufagenin and ouabain with ATPase are practically the same (~0.5 | M), but the energy profile for these two reactions is dramatically different. Ouabain binding is enthalpy favorable process, the marinobufagenin binding is entropy driven. Moreover marinobufagenin binds to Na,K-ATPase with two type of sites characterized by Kd 0.5 |M with stoichiometry 1 : 1 mol/mol enzyme (first type) and 4 : 1 mol/mol, Kd 4 |M (second type). The data demonstrate that formation of complex ouabain or marinobufeganin with Na,K-ATPase provides different enzyme conformations.
Study was supported by RFBR grant № 12-04-00103a.
References
1. Akimova, O.A, Bagrov, A.Y., Lopina, O.D., et al. J. Biol. Chem, 2005, 280, pp. 832-839.
2. Steinberg, M., Karlish, S.J. J. Biol. Chem., 1989, 264, pp. 2726-2734.
Бюллетень сибирской медицины, 2013, том 12, № 4, с. 24-68
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