Biochimica et Biophysica Acta (BBA) - Biomembranes
Volume 1828, Issue 2, February 2013, Pages 568-576
Fluorone dyes have binding sites on both cytoplasmic and extracellular domains of Na,K-ATPase
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Graphical abstract
Highlights
► Monitoring of Na,K-ATPase interactions with fluorone dyes by fluorescence methods ► Two binding sites: cytoplasmic ATP-binding pocket and extracellular part of enzyme ► Fluorone dyes are good probes for monitoring of intersubunit interactions. ► 5(6)-Carboxyeosin is the best probe for examination of the ATP-binding site. ► Fluorescein is the most sensitive probe for monitoring E1/E2 conformational change.
Abbreviations
NKA
Na,K-ATPase, sodium pump
SERCA
sarco(endo)plasmic reticulum
C23
cytoplasmic segment connecting the second and third transmembrane helices, other cytoplasmic segments analogously
ATP
adenosine 5′-triphosphate
RB
Rose Bengal
ErB
Erythrosine B
EY
Eosin Y
CE
5(6)-carboxyeosin
Fl
Fluorescein
PMSF
Phenylmethylsulfonyl Fluoride
IPTG
isopropyl β-d-thiogalactoside
DTT
dithiothreitol
SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
TCSPC
time-correlated single photon counting method
IRF
instrument response function
KSV
Stern–Volmer quenching constant
KD
dissociation constants
FITC
fluorescein isothiocyanate
Keywords
Na,K-ATPase
C45 loop
Binding sites
Fluorone dyes
Time-resolved fluorescence
Fluorescence quenching
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