Modulation of pig kidney Na+/K+-ATPase activity by cholesterol: Role of hydration

Cholesterol is known to affect the activity of membrane-hound enzymes, incl uding Na+/K+-ATPase. To gain insight into the mechanism of cholesterol's ef fect, we have used various hydrophobic fluorescent probes which insert into different regions of the membrane bilayer and report an the degree of hydr ation of their environment. Specifially, we have measured the generalized p olarization of Laurdan and the lifetime of DPH and derivatives of DPH inser ted into membranes from pig kidneys enriched in Na+/K+-ATPase. Spectral mea surements were also carried out on these membranes after modification of th eir cholesterol content. The generalized polarization of Laurdan increased with increasing cholesterol, showing an abrupt modification at the native c holesterol content. The fluorescence lifetimes of DPH and the DPH derivativ es were analyzed using a distribution model. The center value of these life time distributions and their widths also changed with increasing cholestero l. One DPH derivative, DPH-PC, showed a minimum value for the lifetime cent er at the native cholesterol concentration, whereas the other derivatives s howed a maximum value for the lifetime center at that cholesterol concentra tion. DPH-PC is known to sense the protein-lipid interface, whereas the oth er derivatives sense the bulk lipid phase. These data suggest that hydratio n at the protein-lipid interface is maximal at the native cholesterol conce ntration as is the enzymatic activity. Hydration at the protein-lipid inter face is therefore proposed to be required for activity. These results are i n agreement with current models of membrane dynamics and thermodynamics of protein function.