Fluorescence competition assay for the assessment of ATP binding to an isolated domain of Na+, K(+)-ATPase
Language English Country Czech Republic Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
14984322
Knihovny.cz E-resources
- MeSH
- Adenosine Triphosphate chemistry metabolism MeSH
- Models, Chemical * MeSH
- Fluorescence MeSH
- Binding, Competitive MeSH
- Sodium-Potassium-Exchanging ATPase chemistry metabolism MeSH
- Protein Structure, Tertiary MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Adenosine Triphosphate MeSH
- Sodium-Potassium-Exchanging ATPase MeSH
An equation allowing estimation of the dissociation constant for binding of a non-fluorescent ligand to the enzyme is presented that is based on the competitive replacement of the ligand by its fluorescent analog. We derived an explicit formula for the probe fluorescence intensity, which is suitable for nonlinear least-squares analysis. We used this formula to evaluate the binding of ATP to the large cytoplasmic loop of Na+,K(+)-ATPase. The estimated value of KD (6.2+/- 0.7 mM) is comparable with the results from other laboratories for similar constructs obtained by a different method.
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