Time resolution of fluorescence changes observed in titrations of fluorescein 5'-isothiocyanate-modified Na,K-ATPase with monovalent cations.
Equilibrium fluorometric titrations of fluorescein 5'-isothiocyanate-modified Na,K-ATPase with cations have usually been interpreted by assuming that an enhancement reports the conformational change from E2 to E1. We report time resolution of the fluorescence change into three phases when fluorophore-modified enzyme is mixed with the chloride salt of either sodium or choline in a stopped-flow instrument. The first phase is an increase in fluorescence within the dead time of the instrument that is also observed when fluorescein 5'-isothiocyanate (FITC) reacted with lysine is substituted for fluorescein-labeled enzyme. The other two phases occur on millisecond and second time scales. Three phases are also observed when fluorophore-modified enzyme preincubated in KCl is mixed with NaCl, but in this case the slowest phase is absent when choline chloride replaces NaCl. The two faster effects in phases one and two can be eliminated either by controlling the ionic strength or by anti-fluorescein antibody. Labeling the enzyme with fluorescein 5'-isothiocyanate in the presence of its substrate, adenosine 5'-triphosphate, practically eliminates the slowest effect. These results demonstrate that fluorescein reports three events that occur on three different time scales. The fastest phase reports the ionic strength jump of unbound fluorophore. The intermediate phase reports the ionic strength jump of fluorescein at "antibody-accessible" sites [Abbott, A. J., Amler, E., & Ball, W. J., Jr. (1991) Biochemistry 30, 1692-1701]. Only the slowest phase reports the enzyme conformational change implicated in transport.(ABSTRACT TRUNCATED AT 250 WORDS)
Lin
S H
SH
Department of Medicine, Los Angeles School of Medicine, University of California.
Faller
L D
LD
D000818:Animals; D002414:Cations, Monovalent; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D007700:Kinetics; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013552:Swine
D002414:Cations, Monovalent; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate