The sided action of Na+ on reconstituted shark Na+/K+-ATPase engaged in Na+-Na+ exchange accompanied by ATP hydrolysis. II. Transmembrane allosteric effects on Na+ affinity.

The objective of the present investigation was to characterize the ATP-dependent Na+-Na+ exchange, with respect to cation sensitivity on the two aspects of the Na+/K+-pump protein. In order to accomplish this, we used Na+/K+-ATPase reconstituted with known orientation in the proteoliposomes. Activation by cytoplasmic Na+ shows cooperative interaction between three sites. The apparent intrinsic site constants displayed transmembrane dependence on the extracellular Na+ concentration. However, the apparent K0.5 for cytoplasmic Na+ is independent of the extracellular Na+ concentration. The activation by extracellular Na+ at a fixed cytoplasmic Na+ concentration is biphasic with a component which saturates at a concentration of about 1-2 mM extracellular Na+, a plateau phase up to 20 mM, and another component which tends to saturate at about 80 mM followed by a slight deactivation at higher concentrations of Na+. The apparent K0.5 value for extracellular Na+ is also found to be independent of the Na+ concentration on the opposite side of the membrane. The activation by extracellular Na+ can be explained by the negative cooperativity in the binding of extracellular Na+, but positive cooperativity in the rate of dephosphorylation of enzyme species with one and three sodium ions bound extracellularly. Na+ bound to E2-PNa has a transmembrane effect on the cooperativity between binding of cytoplasmic Na+, and E2-PNa2 does not dephosphorylate. K0.5/Vm for cytoplasmic as well as for extracellular Na+ decreases with an increase in the trans Na+ concentration in the non-saturating concentration range. The experiments indicate that at a step in the reaction simultaneous binding of extracellular and cytoplasmic Na+ occurs.