Modulation of Na,K-ATPase and Na-ATPase activity by phospholipids and cholesterol. I. Steady-state kinetics

The effects of phospholipid acyl chain length (n(c)), degree of acyl chain saturation, and cholesterol on Na,K-ATPase reconstituted into liposomes of defined lipid composition are described. The optimal acyl chain length of m onounsaturated phosphatidylcholine in the absence of cholesterol was found to be 22 but decreased to 18 in the presence of 40 mol % cholesterol. This indicates that the hydrophobic matching of the lipid bilayer and the transm embrane hydrophobic core of the membrane protein is a crucial parameter in supporting optimal Na,K-ATPase activity. In addition, the increased bilayer order induced by both cholesterol and saturated phospholipids could be imp ortant for the conformational mobility of the Na,K-ATPase changing the dist ribution of conformations. Lipid fluidity was important for several paramet ers of reconstitution, e.g., the amount of protein inserted and the orienta tion in the liposomes. The temperature dependence of the Na,K-ATPase as wel l of the Na-ATPase reactions depends both on phospholipid acyl chain length and on cholesterol. Cholesterol increased significantly both the enthalpy of activation and entropy of activation for Na,K-ATPase activity and Na-ATP ase activity of Na,K-ATPase reconstituted with monounsaturated phospholipid s. In the presence of cholesterol the free energy of activation was minimum at a lipid acyl chain length of 18, the same that supported maximum turnov er. In the case of ATPase reconstituted without cholesterol, the minimum fr ee energy of activation and the maximum turnover both shifted to longer acy l chain lengths of about 22.