The Sodium Pump is Confined in a Phosphoenzyme Form by Lead(II) Ions

Steady-state fluorescence spectroscopy was used to investigate the effect of Pb2+ ions on the Na+,K+-ATPase. Experiments were performed by using the electrochromic styryl dye RH421 (1, 2) to characterize in detail the effect of Pb2+ ions on the Na-pump and to pinpoint the reaction step(s) of the enzymatic cycle at which the heavy-metal ions provoke their action. We recently found that Pb2+ ions completely inhibit enzyme activity at concentrations above 10 µM (KI = 0.5 µM (3)). It is now shown that Pb2+ ions can bind reversibly to the protein and do not affect the Na+ and K+ binding affinities in the E1 and P-E2 conformations of the enzyme. This indicate that Pb2+ binding to the protein does not block the access pathway to ion binding sites. We also found that lead(II) favors binding of one H+ to the P-E2 conformation in the absence of K+. A model scheme is proposed that accounts for the experimental results obtained for backdoor phosphorylation of the enzyme in the presence of Pb2+ ions. In conclusion, we demonstrate that Pb2+ bound to the enzyme stabilizes an E2-type conformation. In particular, under conditions that promote enzyme phosphorylation, Pb2+ ions are able to confine the Na+,K+-ATPase into a phosphorylated E2 state (4). Acknowledgments The authors thank prof. H.-J. Apell for providing them with membrane fragments containing the Na+,K+-ATPase. The MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca) and the Ente Cassa di Risparmio di Firenze, are gratefully acknowledged for financial support. 1. Grinvald, A., et al., Biophys. J. 39(1982), 301-308. 2. Bühler, R., et al., J. Membr. Biol. 121(1991), 141-161. 3. Gramigni, E., et al., Chem. Res. Toxicol. 22(2009), 1699-1704. 4. Bartolommei, G., et al., Biophys. J. doi:10.1016/j.bpj.2010.07.050(2010), in press.