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RH421 binds into the ATP-binding site on the Na+/K+-ATPase
M. Huličiak, V. Bazgier, K. Berka, M. Kubala,
Language English Country Netherlands
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Cell Membrane metabolism MeSH
- Cytoplasm metabolism MeSH
- Fluorescent Dyes metabolism MeSH
- Kinetics MeSH
- Mutagenesis, Site-Directed methods MeSH
- Molecular Docking Simulation MeSH
- Sodium-Potassium-Exchanging ATPase metabolism MeSH
- Tryptophan metabolism MeSH
- Binding Sites MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The Na+/K+-ATPase plays a key role in ion transport across the plasma membrane of all animal cells. The voltage-sensitive styrylpyrimidium dye RH421 has been used in several laboratories for monitoring of Na+/K+-ATPase kinetics. It is known, that RH421 can interact with the enzyme and it can influence its activity at micromolar concentrations, but structural details of this interaction are only poorly understood. Experiments with isolated large cytoplasmic loop (C45) of Na+/K+-ATPase revealed that RH421 can interact with this part of the protein with dissociation constant 1μM. The Trp-to-RH421 FRET performed on six single-tryptophan mutants revealed that RH421 binds directly into the ATP-binding site. This conclusion was further supported by results from molecular docking, site-directed mutagenesis and by competitive experiments using ATP. Experiments with C45/DPPC mixture revealed that RH421 can bind to both C45 and lipids, but only the former interaction was influenced by the presence of ATP.
References provided by Crossref.org
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- $a Huličiak, Miroslav $u Department of Biophysics, Faculty of Science, Palacky University, Slechtitelu 27, 783 41 Olomouc, Czech Republic.
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- $a The Na+/K+-ATPase plays a key role in ion transport across the plasma membrane of all animal cells. The voltage-sensitive styrylpyrimidium dye RH421 has been used in several laboratories for monitoring of Na+/K+-ATPase kinetics. It is known, that RH421 can interact with the enzyme and it can influence its activity at micromolar concentrations, but structural details of this interaction are only poorly understood. Experiments with isolated large cytoplasmic loop (C45) of Na+/K+-ATPase revealed that RH421 can interact with this part of the protein with dissociation constant 1μM. The Trp-to-RH421 FRET performed on six single-tryptophan mutants revealed that RH421 binds directly into the ATP-binding site. This conclusion was further supported by results from molecular docking, site-directed mutagenesis and by competitive experiments using ATP. Experiments with C45/DPPC mixture revealed that RH421 can bind to both C45 and lipids, but only the former interaction was influenced by the presence of ATP.
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- $a Bazgier, Václav $u Department of Physical Chemistry, Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacky University, 17. listopadu 12, 771 46 Olomouc, Czech Republic.
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- $a Kubala, Martin $u Department of Biophysics, Faculty of Science, Palacky University, Slechtitelu 27, 783 41 Olomouc, Czech Republic; Department of Biophysics, Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University, Slechtitelu 27, 783 41 Olomouc, Czech Republic. Electronic address: martin.kubala@upol.cz.
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