Binding of Eosin-Y to the Na+/K+-ATPase containing occluded Rb+"

MÓNICA MONTES; RODOLFO GONZÁLEZ LEBRERO; SERGIO KAUFMAN; PATRICIO GARRAHAN; ROLANDO ROSSI

Buenos Aires

Workshop; First Mercosur Workshop on biomembranes; 2000

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FACULTAD DE FARMACIA Y BIOQUÍMCA (UBA) BUENOS AIRES - ARGENTINA email:mmontes@qb.ffyb.uba.ar Introduction Eukaryotic cells have Na+/K+-pumps (Na+/K+-ATPase) in their membranes, which actively transport Na+ ions out and K+ ions into the cell. The pumping requires energy, and the enzyme is an energy transducer, which converts chemical energy from the hydrolysis of ATP into a difference of the electrochemical potential of the transported cations across the cell membrane. The transport cycle occurs through a set of kinetically distinguishable conformational states of the enzyme, one with high affinity for Na+, E1, and the other with high affinity for K+, E2. Some of these states are phosphorylated (E1P and E2P) and some are able to hold the transported cation within the enzyme structure. The latter are known as occluded states, and it is assumed that Na+ and K+ remain occluded in E1P and in E2¸respectively. Conformational changes have been studied in Na+/K+-ATPase using Eosin-Y (eosin) (Skou and Esmann, 1981). In the presence of Na+, eosin binds non-covalently to the enzyme with a high affinity (KD = 0.25-0.5 µM) leading to an increase in its fluorescence. Conversely, in the presence of K+ eosin binds with low affinity and this phenomenon is not reflected by an increase in fluorescence, at least, not at the low concentrations of eosin which can be tested (Esmann and Fedosova, 1997). According to these features, eosin is a probe of the E1 form of the enzyme. In our laboratory, we have developed a method to accurately measure occluded cations, and applied it to the measurement of occlusion of the K+-congener rubidium, thus presumably testing the E2 form of the enzyme. The aim of this work is a comparative characterization of the kinetics of the E1 Ç E2 conformational transition, using both eosin-fluorescence and rubidium-occlusion experiments. As eosin binds non-covalently to the pump, this characterization requires a detailed knowledge of the interaction between the pump, the eosin and the other ligands involved in the conformational transition, i.e. Na+, K+, ATP and Mg2+. The results presented here intend to characterize the effects of eosin on the equilibrium between free and occluded Rb+ (K+) and on the kinetics of release of this cation from the occluded state. Some of the Rb-occlusion measurements presented here were compared to preliminary results obtained from eosin fluorescence experiments, performed under similar equilibrium conditions. Methods Experiments were performed at 25ºC in media containing 25 mM Imidazol pH=7.4 (25ºC), 0.25 mM EDTA and the concentrations of enzyme, eosin and Rb+ indicated in the figure legends. Enzyme: Na+/K+-ATPase was a partially purified preparation obtained from the outer medulla of pig kidney by the procedure described by Jensen et al. (1984). Determination of Rb+ occlusion: We used the procedure described in Rossi et al. (1999). Fluorescence : Measurements of changes in eosin fluorescence were determined using an Aminco-Bowman fluorometer. Eosin fluorescence was measured according to Skou and Esmann (1988). Media containing enzyme and eosin were kept in the dark throughout the experiments. Results and Discussion We determined the amount of occluded Rb+ (Rbocc) in equilibrium conditions within a broad range of eosin (0-550 µM) and rubidium (1-150 µM) concentrations. Results are shown in Figure 1, as a function of the concentration of eosin. Figure 1: Effect of Eosin-Y on Rb+ occlusion in equilibrium conditions. Media contained either1 (æ),10 (ç), 50 (ô,) or 150 (õ) µM of Rb+. The enzyme concentration was 30 µg/ml. To ensure equilibrium conditions, the incubation times were at least 15 minutes. It can be seen that, at a given [Rb+], the amount of occluded Rb+ decreased asymptotically with increasing [eosin] toward a level whose value is different from zero. The results were analyzed by non-linear regression using the following equation:       (1) where N0/D0 and N2 are the values that takes Rbocc in equation (1) in the absence of eosin and at [eosin] tending to infinity, respectively. Equation (1) gave a better fit to the results than a single decreasing hyperbola, which could indicate the presence of more than one class of binding sites for eosin in the enzyme. The best fitting values for N0/D0 and N2 were plotted as a function of [Rb+] (Figure 2). Figure 2: Rbocc calculated from equation (1) for [eosin] = 0, N0/D0(ç) and [eosin]®¥, N2, (æ) as a function of [Rb+]. The values, ± 1 standard error (vertical bars), were obtained by non-linear regression analysis of the results in Figure 1. It can be seen that N2, increases with [Rb+] along a curve that tends to the same value as that of N0/D0 but with much less affinity. These results suggest that eosin-bound enzyme allows Rb+ to become occluded, although with much less affinity for the cation. As Rb+ occlusion is assumed to occur in the E2 form of the enzyme, this means that eosin would be also capable to bind to this conformer, as suggested by Skou and Esmann (1981). We performed equilibrium experiments measuring the fluorescence change of eosin in media with various concentrations of Rb+ and eosin (data not shown). The results seem to agree with the occlusion results, since at a given [eosin], the concentration of Rb+ necessary for half-maximum effect tended to increase with [eosin]. A second source of evidence of the binding of eosin to E2 was obtained from experiments measuring the release of 86Rb+ from the occluded state of the enzyme after a 20 fold isotopic dilution of the radioactive cation (Figure 3). It was observed that the presence of eosin in the dilution medium significantly increased the rate coefficients of 86Rb+ release. This effect of eosin is in agreement with its ability to bind to the ATP sites of the enzyme. Results obtained from eosin-fluorescence experiments by Skou and Esmann (1981) showed that ATP was able to compete with eosin for the binding to the Na+ (E1) form of the enzyme. Although with some reticence, the authors also suggested the existence of a low affinity binding of eosin to the K+ (E2) form of the enzyme. Our results provide a robust evidence for their suspicion.   Figure 3: Time course for the release of 86-Rb+ from occluded Rb+ with 550 µM (ç) and without (æ) eosin. Final concentration of Rb+ was 100 µM. References Esmann, M. and N.U. Fedosova. Ann. N. Y. Acad. Sci., 834, 310-321 (1997). Rossi, R.C., S.B. Kaufman, R.M. González-Lebrero, P.J. Schwarzbaum, J.G. Norby, and P.J. Garrahan. J. Biol. Chem., 274, 20779-20790 (1999). Rossi R.C., S.B. Kaufman., R. González Lebrero, J.G. Norby., and P.J. Garrahan. Anal. Biochem., 270,276-285(1999). Skou, J.C. and M. Esmann. Biochim. Biophys. Acta, 601, 2386-402 (1980). Skou, J.C.and M. Esmann. Biochim. Biophys. Acta, 647, 232-240 (1981). Jensen, J., Norby, J.G., and Ottolengui, P. J. Physiol. 346, 219-241(1984). Skou, J.C. and M. Esmann Methods Enzymol., 156, 278-281 (1988).     [1] This work was supported by grants from ANPCYT, CONICET and Universidad de Buenos Aires, Argentina. [2] Corresponding author: R.C.Rossi - rcr@qb.ffyb.uba.ar