Conformational transitions of detergent-solubilized Na,K-ATPase are conveniently monitored by the fluorescent probe 6-carboxy-eosin

Abstract

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 μM in 20 mM NaCl at 6 °C (pH 7.0) for Na,K-ATPase solubilized in C 12E 8. It is shown that the slow conformational change from E 2 (in K +) to E 1 (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 E 1 to E 2 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.