Inhibitory effect of Pb2+ on the transport cycle of the Na,K-ATPase

The effect of Pb2+ 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 Pb2+ concentrations to investigate the effect of Pb2+ 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 Pb2+ concentration. Our results indicate that Pb2+ 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 microM. To explain the inhibitory effect of Pb2+ on the Na+,K+-ATPase, we propose that Pb2+ may interfere with the hydrolytic cleavage of the phosphorylated intermediate E2P, which occurs in the K+-related branch of the pump cycle.