Modulation of Na,K-ATPase by phospholipids and cholesterol. II. Steady-state and presteady-state kinetics.

The effects of phospholipid acyl chain length (n(c)) and cholesterol on several partial reactions of Na,K-ATPase reconstituted into liposomes of defined lipid composition are described. This regards the E(1)/E(2) equilibrium, the phosphoenzyme level, and the K(+)-deocclusion reaction. In addition, the lipid effects on some steady-state properties were investigated. Finally, the effects of cholesterol on the temperature sensitivity of the phosphorylation and spontaneous dephosphorylation reactions were investigated. The fatty acid and cholesterol composition of the native Na,K-ATPase membrane preparation showed a remarkable similarity to the lipid composition known to support maximum hydrolytic capacity as determined from in vitro experiments. The main rate-determining step of the Na,K-ATPase reaction, the E(2) --> E(1) reaction, as well as several other partial reactions were accelerated by cholesterol. This regards the phosphorylation by ATP as well as the E(1) - P --> E(2)-P reaction. Moreover, cholesterol shifted the E(1)/E(2) equilibrium toward the E(1) conformation and increased the K(+)-deocclusion rate. Finally, cholesterol significantly affected the temperature sensitivity of the spontaneous dephosphorylation reaction and the phosphorylation by ATP. The effects of cholesterol were not completely equivalent to those induced by increasing the phospholipid acyl chain length, indicating that the cholesterol effects are not entirely caused by increasing the hydrophobic bilayer thickness, which indicates an additional mechanism of action on the Na,K-ATPase.