Conformational transitions of detergent-solubilized Na,K-ATPase are conveniently monitored by the fluorescent probe 6-carboxy-eosin.
6-carboxy-eosin is introduced as a sensitive, non-covalently bound fluorescent probe for monitoring conformational changes in detergent-solubilized Na,K-ATPase. The dissociation constant for 6-carboxy-eosin is about 0.1 microM in 20 mM NaCl at 6 degrees C (pH 7.0) for Na,K-ATPase solubilized in C12E8. It is shown that the slow conformational change from E2 (in K+) to E1 (in Na+) is 4-fold more rapid in the solubilized state than in the membrane-bound state, both for shark rectal gland and pig kidney Na,K-ATPase. The rate of the E1 to E2 transition is rapid and of the same order of magnitude both for the membrane-bound and the solubilized enzyme. All conformational transitions are considerably slower for pig kidney enzyme than for shark enzyme, both in the membrane-bound and in the solubilized state.
Esmann
M
M
Institute of Biophysics, University of Aarhus, Denmark.
D000818:Animals; D003902:Detergents / Q000494:pharmacology; D004284:Dogfish; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D011487:Protein Conformation; D012007:Rectum / Q000187:drug effects / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D012995:Solubility; D013552:Swine
D003902:Detergents; D005452:Fluoresceins; D005456:Fluorescent Dyes; C024098:6-carboxyfluorescein; D000254:Sodium-Potassium-Exchanging ATPase