Phosphorylation by P i of the Na,K-ATPase from rabbit kidney in the absence of Na + ions but in the presence of Mg 2+ ions has been studied. In the absence of K + ions, unphosphorylated and phosphorylated states induce different fluorescence levels in the membrane-bound styryl dye RH421, and hence transitions between the two states were monitored. Transient kinetic studies of phosphorylation were initiated by manual addition of P i or by photochemical release of P i from 1-(2-nitrophenyl)ethyl phosphate (caged P i ) using laser flash photolysis at 308 nm. Equilibrium studies of phosphorylation showed that the apparent K m for P i was 23.0 ( 0.3 µM (mean ( sem) at pH 7.1 and 21°C. The dye fluorescence increased in a biphasic manner on addition of 500 µM P i to the enzyme: a rapid phase (t 1/2 < 1 s) and a slower exponential phase at 0.059 ( 0.003 s -1 . The rate of the rapid phase was studied by fast concentration-jump experiments and exhibited first-order kinetics in P i up to 60 µM. Fluorescence records vs time were exponential, and a plot of the rate constant versus [P i ] had a slope of 1.47 × 10 5 M -1 s -1 and ordinate ([P i ] ) 0) intercept of 3.1 s -1 . Addition of 50 mM NaCl to the phosphorylated enzyme induced an exponential decay in the dye fluorescence from which a rate constant of 0.10 ( 0.005 s -1 was determined. These data were interpreted in terms of transformations between conformational states E 1 and E 2 , and the phosphorylated state P-E 2 defined in the Post-Albers mechanism of the Na,K-ATPase [Läuger, P., (1991) Electrogenic Ion Pumps, Sinauer Associates Inc., Sunderland, MA] ass -1 , and k -2 ) 3.1 s -1 . The RH421 fluorescence of state P-E 2 was studied over the pH range 6-8.5. Fluorescence was greatest at pH 8.5 and lowest at pH 6.0 in a simple binding isotherm with pK 7.5. The apparent K m for P i rose cooperatively with increasing pH (pK a 8.6 and a Hill coefficient of 2). Therefore in the absence of monovalent metal ions, occupation of the cation (K + ) binding sites by protons promotes phosphorylation by P i .

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)py ridinium 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 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 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 (E1ATP(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<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively 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) microM. 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/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.

Abstract.A fluorescence method was adapted to investigate active ion transport in membrane preparations of the SR-Ca-ATPase. The styryl dye RH421 previously used to investigate the Na,K-ATPase was replaced by an analogue, 2BITC, to obtain optimized fluorescence changes upon substrate-induced partial reactions. Assuming changes of the local electric field to be the source of fluorescence changes that are produced by uptake/ release or by movement of ions inside the protein, 2BITC allowed the determination of electrogenic partial reactions in the pump cycle. It was found that Ca 2+ binding on the cytoplasmic and on the lumenal side of the pump is electrogenic while phosphorylation and conformational transition showed only minor electrogenicity. Ca 2+ equilibrium titration experiments at pH 7.2 in the two major conformations of the protein indicated cooperative binding of two Ca 2+ ions in state E 1 with an apparent half-saturation concentration, K M of 600 nM. In state P-E 2 two K M values, 5 M and 2.2 mM, were determined and are in fair agreement with published data. From Ca 2+ titrations in buffers with various pH and from pH titrations in P-E 2 , it could be demonstrated that H + binding is electrogenic and that Ca 2+ and H + compete for the same binding site(s). Tharpsigargin-induced inhibition of the Ca-ATPase led to a state with a specific fluorescence level comparable to that of state E 1 with unoccupied ion sites, independent of the buffer composition.

Abstract. A family of¯uorescent styryl dyes was synthesized to apply them as probes that monitor the ion-translocating activity of the Na,K-ATPase and the SR Ca-ATPase, similar to the widely used dye RH421. All dyes had the same chromophore but they di ered in the length of the spacer between chromophore and polar head, an isothiocyanate group, and in the lengths of the two identical acyl chains, which form the tail of the dye molecules. A number of substrate-dependent partial reactions of both P-type ATPases a ected the¯uorescence intensity, and the magnitude of the¯uorescence changes was used to characterize the usefulness of the dyes for further application. The experimental results indicate that electrochromy is the major mechanism of these dyes. While in the case of the Na,K-ATPase a single dye, 5QITC, showed larger¯uorescence changes than all others, in the case of the SR Ca-ATPase all dyes tested were almost equal in their¯uorescence responses. This prominent di erence is interpreted as a hint that the position of the ion binding sites in both ion pumps may di er signi®cantly despite their otherwise closely related structural features. Quench experiments with spin-labeled lipids in various positions of their fatty acids were used to gain information on the depth of the chromophore of the di erent dyes within the membrane dielectric, however, the spatial resolution was so poor that only qualitative information on the position of the chromophore in the lipid phase could be obtained.

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