Na(+) transport, and the E(1)P-E(2)P conformational transition of the Na(+)/K(+)-ATPase.
We have used admittance analysis together with the black lipid membrane technique to analyze electrogenic reactions within the Na(+) branch of the reaction cycle of the Na(+)/K(+)-ATPase. ATP release by flash photolysis of caged ATP induced changes in the admittance of the compound membrane system that are associated with partial reactions of the Na(+)/K(+)-ATPase. Frequency spectra and the Na(+) dependence of the capacitive signal are consistent with an electrogenic or electroneutral E(1)P <--> E(2)P conformational transition which is rate limiting for a faster electrogenic Na(+) dissociation reaction. We determine the relaxation rate of the rate-limiting reaction and the equilibrium constants for both reactions at pH 6.2-8.5. The relaxation rate has a maximum value at pH 7.4 (approximately 320 s(-1)), which drops to acidic (approximately 190 s(-1)) and basic (approximately 110 s(-1)) pH. The E(1)P <--> E(2)P equilibrium is approximately at a midpoint position at pH 6.2 (equilibrium constant approximately 0.8) but moves more to the E(1)P side at basic pH 8.5 (equilibrium constant approximately 0.4). The Na(+) affinity at the extracellular binding site decreases from approximately 900 mM at pH 6.2 to approximately 200 mM at pH 8.5. The results suggest that during Na(+) transport the free energy supplied by the hydrolysis of ATP is mainly used for the generation of a low-affinity extracellular Na(+) discharge site. Ionic strength and lyotropic anions both decrease the relaxation rate. However, while ionic strength does not change the position of the conformational equilibrium E(1)P <--> E(2)P, lyotropic anions shift it to E(1)P.
Babes
A
A
Department of Physiology and Biophysics, Faculty of Biology, University of Bucharest, Bucharest, Romania.
Fendler
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D004073:Digitoxigenin / Q000494:pharmacology; D004563:Electrochemistry; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D017136:Ion Transport; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008954:Models, Biological; D008985:Monensin / Q000494:pharmacology; D010782:Photolysis; D011487:Protein Conformation; D015398:Signal Transduction / Q000187:drug effects; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D013552:Swine; D013816:Thermodynamics
D007476:Ionophores; D008051:Lipid Bilayers; D004073:Digitoxigenin; D000255:Adenosine Triphosphate; D008985:Monensin; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase