Membrane receptor calorimetry: cardiac glycoside interaction with Na,K-ATPase

Citation
E. Grell et al., Membrane receptor calorimetry: cardiac glycoside interaction with Na,K-ATPase, THERMOC ACT, 380(2), 2001, pp. 245-254
Citations number
30
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Spectroscopy /Instrumentation/Analytical Sciences
Journal title
THERMOCHIMICA ACTA
ISSN journal
0040-6031 → ACNP
Volume
380
Issue
2
Year of publication
2001
Pages
245 - 254
Database
ISI
SICI code
0040-6031(200112)380:2<245:MRCCGI>2.0.ZU;2-L
Abstract
The receptor-ligand interaction between the cardiac glycoside Ouabain and p urified, membrane-bound as well as micellar Na,K-ATPase is investigated. Ca lorimetric titrations are carried out with micromolar concentrations of the phosphorylated protein in the presence of Mg2+. The measured heat changes provide evidence for an exothermic, high affinity and specific receptor bin ding process as well as for a low affinity, nonspecific binding to the lipi d part of the nanoparticulate membrane fragments. The degree of lipid bindi ng markedly depends on the lipid composition of the tissue. The measured ti me course of the heat chan-e resulting from specific binding to the recepto r site is unusually slow and is limited by the binding kinetics of the liga nd. A course estimation of the Ouabain binding kinetics leads to a rate con stant around 10(4) mol(-1) 1 s(-1). Receptor binding is characterized by af finities ranging between 10(7) and 10(8) mol(-1) 1, DeltaH values around -9 5 kJ mol(-1) and DeltaS values of about - 130 J K-1 mol(-1) at 25 degreesC. The enthalpic contribution is assumed to be mainly due to hydrogen bond fo rmations between the ligand and the receptor site whereas the large, negati ve entropy change may be attributed to an increased interaction between wat er and the protein as a consequence of a conformational transition. The eva luation of the titrations provides stoichiometric coefficients around 0.55, which implies that only about 50-60% of the Na,K-ATPase protomers are capa ble to bind the cardiotonic steroid. This result is consistent with radioac tive phosphorylation studies and appears to be a typical feature of kidney- type Na,K-ATPase preparations. Possible implications of this finding are di scussed. As a general result, this study demonstrates how simple and suitab le calorimetric titrations with micromolar protein concentrations can be fo r the purpose of a quantitative characterization of a receptor in nanoparti culate membrane systems. (C) 2001 Elsevier Science B.V All rights reserved.