Publikation: Kinetics of Na+-dependent conformational changes of rabbit kidney Na+,K+-ATPase
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The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)pyridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/1 155 s1 for 5-IAF-labeled enzyme and 1/1 200 s1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24°C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/2 = 30-45 s1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P E2). The Na+ concentration dependence of 1/1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/1 was 8.0 (± 0.7) µM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/1 observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.
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CLARKE, Ronald J., David J. KANE, Hans-Jürgen APELL, Milena ROUDNA, Ernst BAMBERG, 1998. Kinetics of Na+-dependent conformational changes of rabbit kidney Na+,K+-ATPase. In: Biophysical Journal. 1998, 75(3), pp. 1340-1353. ISSN 0006-3495. Available under: doi: 10.1016/S0006-3495(98)74052-4BibTex
@article{Clarke1998Kinet-7209, year={1998}, doi={10.1016/S0006-3495(98)74052-4}, title={Kinetics of Na+-dependent conformational changes of rabbit kidney Na+,K+-ATPase}, number={3}, volume={75}, issn={0006-3495}, journal={Biophysical Journal}, pages={1340--1353}, author={Clarke, Ronald J. and Kane, David J. and Apell, Hans-Jürgen and Roudna, Milena and Bamberg, Ernst} }
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