The effect of Pb(2+) on the transport cycle of the Na(+),K(+)-ATPase was characterized in detail at a molecular level by combining electrical and biochemical measurements. Electrical measurements were performed by adsorbing purified membrane fragments containing Na(+),K(+)-ATPase on a solid-supported membrane. Upon adsorption, the Na(+),K(+)-ATPase was activated by carrying out concentration jumps of different activating substrates, for example, Na(+) and ATP. Charge movements following Na(+),K(+)-ATPase activation were measured in the presence of various Pb(2+) concentrations to investigate the effect of Pb(2+) on different ion translocating steps of the pump cycle. These charge measurements were then compared to biochemical measurements of ATPase activity in the presence of increasing Pb(2+) concentration. Our results indicate that Pb(2+) inhibits cycling of the enzyme, but it does not affect cytoplasmic Na(+) binding and release of Na(+) ions at the extracellular side at concentrations below 10 muM. To explain the inhibitory effect of Pb(2+) on the Na(+),K(+)-ATPase, we propose that Pb(2+) may interfere with the hydrolytic cleavage of the phosphorylated intermediate E(2)P, which occurs in the K(+)-related branch of the pump cycle.

No abstract

Metals are one of the major classes of environmental contaminants and raise concerns for their adverse effects on ecosystems. Ants are good candidates as bioindicators for metal contamination assessment; previous studies indeed showed that ants are able to selectively accumulate some metals within their tissues. Available works provide only whole-body burdens of these contaminants, with scarce information on the fine-scale localisation in tissues and organs, although this information is important to better understand the behaviour of metals in living organisms and to clarify their effects in ecosystems. At the Florence external scanning microbeam, we are carrying on a Particle Induced X-ray Emission (PIXE) study on a common ant species sampled from sites with different environmental metal availabilities. Measurements were carried out on resin-embedded, self-standing sections for a direct localisation of metal deposits and an easy determination of their content. The combined use of the PIXE and the external scanning microbeam made it possible to map element distributions with good spatial resolution and sensitivity, restricting quantitative analyses to the metal accumulation regions. To determine in which tissues/organs metals concentrated, we compared PIXE maps with histological images on sections contiguous to the analysed slices. Measurements in the external set-up allowed us to avoid sample damaging. Differences in metal concentrations in ants from different sites resulted from quantitative PIXE analyses.

The effect of Pb(2+) 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 Pb(2+) ions can bind reversibly to the protein and do not affect the Na(+) and K(+) binding affinities in the E(1) and P-E(2) conformations of the enzyme. The pH titrations indicate that lead(II) favors binding of one H(+) to the P-E(2) 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 Pb(2+) ions. Taken together, our results clearly indicate that Pb(2+) bound to the enzyme stabilizes an E(2)-type conformation. In particular, under conditions that promote enzyme phosphorylation, Pb(2+) ions are able to confine the Na(+),K(+)-ATPase into a phosphorylated E(2) state.

a protonatable side chain in E2P-like conformations might allow shuttling of protons across the membrane. We therefore evaluated the role of three key carboxylates, E336 (TM4), E788 (TM5), and D935 (TM8), by mutating C113Y Xenopus pumps. Outward Na/K-pump current was practically abolished in E336Q(C113Y) or E336C(C113Y) pumps, but nonstoichiometric inward current at zero Ko and Nao (replaced by TMAo) was little altered, and was greatly augmented in high Nao. E788C(C113Y) pumps, on the other hand, like parent C113Y pumps, generated robust stoichiometric outward Na/K transport currents (voltage dependent in Nao, but not in TMAo), and little nonstoichiometric inward current in high Nao; but nonstoichiometric current in TMAo was dimin-ished~3-fold compared to parent C113Y pumps. In contrast, D935N(C113Y) pumps generated stoichiometric outward Na/K transport currents with altered voltage dependence that was similar in Nao or in TMAo, but inward nonstoichiometric current was nearly absent both in Nao and in TMAo, and was not augmented by lowering pH to 6 either in Nao and in TMAo. The D935 carboxylate thus seems uniquely required for the nonstoichiometric inward flow of protons through the Na/K-pump. [NIH HL36783].