Eosin fluorescence changes during Rb+ occlusion in the Na+/K(+)-ATPase.
We used suspensions of partially purified Na(+)/K(+)-ATPase from pig kidney to compare the effects of Rb(+), as a K(+) congener, on the time course and on the equilibrium values of eosin fluorescence and of Rb(+) occlusion. Both sets of data were collected under identical conditions in the same enzyme preparations. The incubation media lacked ATP so that all changes led to an equilibrium distribution between 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 decreased and occlusion increased along rectangular hyperbolas with similar half-maximal values. The time courses of attainment of equilibrium showed an initial phase which was so quick as to fall below the time resolution of our rapid-mixing apparatus. This phase was followed by the sum of at least two exponential functions of time. In the case of fluorescence the fast exponential term accounted for a larger fraction of the time course than in the case of occlusion. Comparison between experimental and simulated results suggests that fluorescence changes express a process that is coupled to Rb(+) occlusion but that is completed before occlusion reaches equilibrium.
Montes
Mónica R
MR
Instituto de Química y Fisicoquímica Biológicas, Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, C1113AAD Buenos Aires, Argentina.
González-Lebrero
Rodolfo M
RM
Garrahan
Patricio J
PJ
Rossi
Rolando C
RC
D000465:Algorithms; D000818:Animals; D004801:Eosine Yellowish-(YS) / Q000737:chemistry* / Q000378:metabolism; D005453:Fluorescence; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D012413:Rubidium / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013552:Swine
D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D004801:Eosine Yellowish-(YS)