Kinetics of Na+-dependent conformational changes of rabbit kidney Na+,K+-ATPase

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)pyridium 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/tau(1) approximate to 155 s(-1) for 5-IAF-labeled enzyme and 1/tau(1) approximate to 200 s(-1) for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E(1)ATP(Na+)(3) --> E2P(Na+)(3) + ADP). The smaller amplitude slow phase, 1/tau(2) = 30-45 s(-1), is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P double left right arrow E-2). The Na+ concentration dependence of 1/tau(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 tau(1), was 8.0 (+/- 0.7) mu M. It was found that P-3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau(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.