Kinetic Characterization of Intermediates During Na-ATPase and Na,K-ATPase Activity Using RH421 and Rb+ Occlusion

P. G. SCHVARTZ; J. L. E. MONTI; R. M. GONZÁLEZ-LEBRERO; S. B. KAUFMAN; P. J. GARRAHAN; R. C. ROSSI

MBL, Woods Hole, Massachusetts, USA

Congreso; 11th International ATPase Conference and 59th Annual Meeting and Symposium of the Society of General Physiologists; 2005

The Society of General Physiologists

Employment of the fluorescent probe RH421 for studying intermediate states of the sodium pump reveals three main levels of fluorescence, the lowest corresponding to the states having 3 Na+ bound, the highest to E2P, and the intermediate level to the states with 2 K+ bound or occluded (Sturmer et al.1991, J.Membr.Biol 121(2):141-61). On the basis of this interpretation, quantification of the intermediate states by measurements of fluorescence changes should be possible. This work was aimed to investigate the ability of the fluorescence probe RH421 to kinetically characterize the intermediates E2P and E2(Rb2) as well as the ATPase activity. With this aim, we followed the time courses of the change of fluorescence of the enzyme performing Na-ATPase activity, until ATP was completely hydrolyzed. Media contained 150 mM NaCl, 0.5 mM free Mg2+, and ATP from 0.1 to 25.6 mM, at 25°C and 25 mM Imidazole-HCl, pH 7.4. Results from these experiments allowed calculating the Na-ATPase activity, being the value of KM for ATP very similar to those found in the literature (Kane et al., 1997, Biochemistry 36:13406-13420). In another series of experiments where different concentrations of Rb+ were added to the media, we measured in parallel experiments the time courses of fluorescence change and of 86Rb+ occlusion. We found that the steady-state levels of both measurements show similar affinities for [Rb+] and that the increasing parts of the time courses at saturating [Rb+] have the same rate coefficients. A model based on that of Albers and Post was fitted to the experimental results. The model allowed simulating the time courses of both the fluorescence change and of Rb+ occlusion after setting a high-fluorescence coefficient value for E2P and a lower fluorescence coefficient for the Rb+-bound states.