Fluoresceinyl-ethylenediamine-ouabain detects an acidic environment in the cardiac glycoside binding site of Na+/K+-ATPase.
To probe the pH value in the microenvironment of the cardiac glycoside-binding site of Na+/K+-ATPase, pH-sensitive fluorescent derivatives of ouabain were synthesized. The fluoresceinyl derivative of ethylenediamino-ouabain (FEDO) had a pKs of 6.0 and showed a H+-dependent fluorescence change, when its ratio of excitation at 490 nm/450 nm was recorded at 530 nm. Binding of FEDO inactivated Na+/K+-ATPase at 37 degrees C and pH 7.25 in a slow time-dependent process under the conditions of backdoor phosphorylation with k(on) of 891 s(-1) M(-1). The complex dissociated with k(on) of 0.35 x 10(-3) s(-1) resulting in a Kd value of 0.4 microM for the FEDO x enzyme complex. Binding of FEDO was associated with a decrease of the excitatory fluorescence ratio at 490 nm/450 nm which could be used to convert this change into a pH value. A pH value of 5.1 +/- 0.2 was calculated to exist in the microenvironment of the FEDO x enzyme complex. This pH value was independent of the pH of the incubation medium used to form the FEDO x enzyme complex. Analysis of the accessibility of the fluorophore in the FEDO x enzyme complex to the dynamic quencher potassium iodide detected a decrease of the Stern-Volmer constant from 6.2 mM(-1) (free FEDO) to 1.5 mM(-1) (FEDO x enzyme complex) indicating thereby a limited accessibility of the fluorophore to anions. Analysis of the microenvironment of the fluorescein residue of the FEDO x enzyme complex by measurements of the anisotropy and the fluorescence half-life time revealed that both processes differed significantly when H2O was replaced by D2O. We conclude, therefore, that a pH of 5.1 +/- 0.2 exists in the vicinity of ouabain that is hidden in the depth of the receptor site when the ouabain receptor complex has been formed.
Brinkmann
K
K
Institute of Biochemistry and Endocrinology, Justus-Liebig-University Giessen, Germany.
Linnertz
H
H
Amler
E
E
Lanz
E
E
Herman
P
P
Schoner
W
W
D000818:Animals; D001665:Binding Sites; D002301:Cardiac Glycosides / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D015394:Molecular Structure; D010042:Ouabain / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D012413:Rubidium / Q000493:pharmacokinetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D002301:Cardiac Glycosides; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; C109048:fluoresceinyl ethylenediamine-ouabain; C109049:rhodol green-ethylenediamine-ouabain; C109050:seminaphthofluoresceinyl ethylenediamine-ouabain; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium