Eosin fluorescence changes and Rb+ occlusion during the E1 to E2 transition in the Na/K-ATPase

MÓNICA R MONTES, RODOLFO M GONZÁLEZ-LEBRERO, PATRICIO J GARRAHAN, AND ROLANDO C ROSSI.

BIOCHEMISTRY

Año: 2007 vol. 43 p. 13093 - 13100

0006-2960

We used suspensions of partially purified Na+/K+-ATPase from pig kidney to compare theeffects of Rb+, as a K+ congener, on the time course and on the equilibrium values of eosin fluorescenceand of Rb+ occlusion. Both sets of data were collected under identical conditions in the same enzymepreparations. The incubation media lacked ATP so that all changes led to an equilibrium distributionbetween enzyme conformers with and without bound eosin and with and without bound or occluded Rb+.Results showed that as Rb+ concentration was increased, the equilibrium value of fluorescence decreasedand occlusion increased along rectangular hyperbolas with similar half-maximal values. The time coursesof attainment of equilibrium showed an initial phase which was so quick as to fall below the time resolutionof our rapid-mixing apparatus. This phase was followed by the sum of at least two exponential functionsof time. In the case of fluorescence the fast exponential term accounted for a larger fraction of the timecourse than in the case of occlusion. Comparison between experimental and simulated results suggeststhat fluorescence changes express a process that is coupled to Rb+ occlusion but that is completed beforeocclusion reaches equilibrium.