ИСТИНА

Experimental data demonstrate that Na,K-ATPase is receptor for cardiotonic steroids (CTS) which can activate signal cascades without pump inhibition. It may be due to the change of Na,K-ATPase conformation that can result in the binding to the enzyme and consequent activation of proteins initiating signal cascades. 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 M1. Using purified Na,K-ATPase from duck salt gland and rabbit kidney we have found that ouabain and marinobufagenin inhibited Na,K-ATPase by similar manner. Simultaneously ouabain and marinobufagenin induce cell death with different values of IC50 (about 0.05 and 5 M respectively). The data suggest that different CTS are able induce different conformations of Na,K-ATPase that can provide the binding to the enzyme different proteins activating signal cascades. Using isothermal titration calorimetry (ITC) we have found that at 25ºC the thermodynamic parameters of marinobufagenin and ouabain binding to Na,K-ATPase are different. Ouabain binding is enthalpy-favorable process with binding constant Ka=2.1×107 M-1; ∆H=-11.8±0.2 kcаl/mol; T∆S=-1.9 kcаl/mol, the marinobufagenin binding is entropy-driven process with binding constant Ka=8.2×105 М-1, ∆H=-2.4±0.2 kcаl/mol; T∆S=5.7 kcаl/mol. Using FITC-labeled Na,K-ATPase we have shown that binding of ouabain and marinobufagenin was described by hyperbolic curves. Maximal change of fluorescence in the presence of ouabain and marinobufagenin was achieved at CTS concentrations that completely inhibited enzyme activity. Fluorescent signal was higher when Na,K-ATPase interacted with saturating concentrations of marinobufagenin. ITC experiments were complemented by ‘‘displacement titrations’’, where marinobufagenin was chased from the Na,K-ATPase-marinobufagenin complex by ouabain. The equilibrium constants for ouabain binding to Na,K-ATPase were estimated from a competitive ligand binding model, and the parameter estimates were virtually the same as those obtained by the direct method. Thus we can propose that both CTS bind to the same site. The data demonstrate that formation of complexes ouabain and marinobufeganin with Na,K-ATPase can provide different enzyme conformations.