Confining the Sodium Pump in a Phosphoenzyme Form: The Effect of Lead(II) Ions

Abstract

AbstractThe effect of Pb2+ ions on the Na+,K+-ATPase was investigated in detail by means of steady-state fluorescence spectroscopy. Experiments were performed by using the electrochromic styryl dye RH421. It is 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. The pH titrations indicate 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. Taken together, our results clearly indicate 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