Transient kinetics and thermodynamics of anthroylouabain binding to Na/K-ATPase.
The Na/K-ATPase is an integral membrane protein enzyme which uses energy derived from hydrolysis of ATP to pump Na+ out of and K+ into the cell. Ouabain belongs to a class of drugs known as cardiac glycosides, which are useful for treating congestive heart failure. Therapeutic value is achieved when these drugs bind to and inhibit the Na/K-ATPase of cardiac muscle. We gain insight into this important interaction by measuring the thermodynamics of the interaction of anthroylouabain (AO), a fluorescent derivative of ouabain, with the Na/K-ATPase. AO has the useful property that its fluorescence intensity is greatly enhanced (approximately 10x) when it binds to the enzyme. Using this enhancement, we measure temperature dependence of transient kinetics for the association and dissociation of AO interacting with membrane fragments of Na/K-ATPase purified from dog kidney. Using a standard Eyring analysis, we find that the overall association of AO with the enzyme is driven by substantial contributions from both enthalpy and entropy, and that in an energy diagram for the association pathway, the free energy change is quite similar to that of ouabain deduced from previously published results [E. Erdmann, W. Schoner, BBA 307 (1973) 386]. However, in the transition state, there are substantial differences for the enthalpy and entropy, presumably due to the presence of the anthracene moiety.
Hellen
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Department of Physics and Astronomy, University of North Carolina at Greensboro 27402, USA.
Yacono
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B
Pratap
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D000818:Animals; D000873:Anthracenes / Q000378:metabolism*; D004285:Dogs; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D010042:Ouabain / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics
D000873:Anthracenes; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010042:Ouabain; C013814:anthroylouabain; D000254:Sodium-Potassium-Exchanging ATPase