E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2
This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb+ deocclusion stimulated by Na + in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb+ and the increas...
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2012
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00052736_v1818_n9_p2087_Montes http://hdl.handle.net/20.500.12110/paper_00052736_v1818_n9_p2087_Montes |
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paper:paper_00052736_v1818_n9_p2087_Montes2023-06-08T14:29:51Z E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2 Conformational transition Na+/K+-ATPase Rb +-deocclusion Vanadate adenosine triphosphatase (potassium sodium) eosin magnesium ion rubidium ion sodium ion vanadic acid amplitude modulator animal tissue article binding affinity conformational transition enzyme inhibitor interaction equilibrium constant fluorescence analysis nonhuman phase transition priority journal Adenosine Triphosphate Animals Biophysics Eosine Yellowish-(YS) Kidney Kinetics Magnesium Models, Biological Protein Binding Protein Conformation Rubidium Sodium-Potassium-Exchanging ATPase Swine Time Factors Vanadates Suidae This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb+ deocclusion stimulated by Na + in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb+ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb+ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb+]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb+-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na +-stimulated Rb+ deocclusion would require the release of at least one Rb+ ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb+ in equilibrium conditions. Results show that, while Mg 2 + decreases the affinity for Rb+, addition of vanadate offsets this effect, increasing the affinity for Rb+. In transient experiments, we investigated the exchange of Rb+ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na+ without significantly affecting the rate of Rb+ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb+ occlusion in the enzyme-vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb+. © 2012 Elsevier B.V. All rights reserved. 2012 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00052736_v1818_n9_p2087_Montes http://hdl.handle.net/20.500.12110/paper_00052736_v1818_n9_p2087_Montes |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Conformational transition Na+/K+-ATPase Rb +-deocclusion Vanadate adenosine triphosphatase (potassium sodium) eosin magnesium ion rubidium ion sodium ion vanadic acid amplitude modulator animal tissue article binding affinity conformational transition enzyme inhibitor interaction equilibrium constant fluorescence analysis nonhuman phase transition priority journal Adenosine Triphosphate Animals Biophysics Eosine Yellowish-(YS) Kidney Kinetics Magnesium Models, Biological Protein Binding Protein Conformation Rubidium Sodium-Potassium-Exchanging ATPase Swine Time Factors Vanadates Suidae |
| spellingShingle |
Conformational transition Na+/K+-ATPase Rb +-deocclusion Vanadate adenosine triphosphatase (potassium sodium) eosin magnesium ion rubidium ion sodium ion vanadic acid amplitude modulator animal tissue article binding affinity conformational transition enzyme inhibitor interaction equilibrium constant fluorescence analysis nonhuman phase transition priority journal Adenosine Triphosphate Animals Biophysics Eosine Yellowish-(YS) Kidney Kinetics Magnesium Models, Biological Protein Binding Protein Conformation Rubidium Sodium-Potassium-Exchanging ATPase Swine Time Factors Vanadates Suidae E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2 |
| topic_facet |
Conformational transition Na+/K+-ATPase Rb +-deocclusion Vanadate adenosine triphosphatase (potassium sodium) eosin magnesium ion rubidium ion sodium ion vanadic acid amplitude modulator animal tissue article binding affinity conformational transition enzyme inhibitor interaction equilibrium constant fluorescence analysis nonhuman phase transition priority journal Adenosine Triphosphate Animals Biophysics Eosine Yellowish-(YS) Kidney Kinetics Magnesium Models, Biological Protein Binding Protein Conformation Rubidium Sodium-Potassium-Exchanging ATPase Swine Time Factors Vanadates Suidae |
| description |
This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb+ deocclusion stimulated by Na + in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb+ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb+ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb+]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb+-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na +-stimulated Rb+ deocclusion would require the release of at least one Rb+ ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb+ in equilibrium conditions. Results show that, while Mg 2 + decreases the affinity for Rb+, addition of vanadate offsets this effect, increasing the affinity for Rb+. In transient experiments, we investigated the exchange of Rb+ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na+ without significantly affecting the rate of Rb+ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb+ occlusion in the enzyme-vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb+. © 2012 Elsevier B.V. All rights reserved. |
| title |
E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2 |
| title_short |
E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2 |
| title_full |
E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2 |
| title_fullStr |
E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2 |
| title_full_unstemmed |
E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2 |
| title_sort |
e2→e1 transition and rb+ release induced by na+ in the na+/k+-atpase. vanadate as a tool to investigate the interaction between rb+ and e2 |
| publishDate |
2012 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00052736_v1818_n9_p2087_Montes http://hdl.handle.net/20.500.12110/paper_00052736_v1818_n9_p2087_Montes |
| _version_ |
1768545354104438784 |