Phospholipid fatty acyl spatial distribution in bovine rod outer segment disk membranes.

The distribution of fatty acids within the phospholipid headgroup classes was investigated as a function of the age/spatial distribution of bovine rod outer segment disk membranes. The disks were separated into subpopulations based upon the cholesterol content in their membranes. Because disk membrane cholesterol content decreases as the disks are apically displaced in the rod outer segment, this separation yields disk subpopulations of different ages and from age-dependent spatial locations within the outer segment. The phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), of each of these subpopulations were separated and the fatty acid composition of each was determined. These data indicated that while most of the fatty acids show little or no change with age/spatial location, some pronounced changes can be observed in certain classes. Within the PC class, 16:0 dramatically decreases with disk age while the 22:6 increases with disk age. While the PE class exhibits some fatty acid changes, they are small. The PS class exhibits no significant changes in fatty acid composition. The PI class which constitutes less than 2% of the total phospholipid exhibits age-related changes in each of the fatty acids which could be measured. Most notable of these is an increase in 20:4 as the disks are apically displaced. These changes indicate a remodeling of the disk membranes which may be related to the phototransduction process or to preparation for eventual disk phagocytosis.

Effect of cholesterol on rhodopsin stability in disk membranes.

The effect of cholesterol on rhodopsin stability has been investigated in intact disk membranes. Because cholesterol readily equilibrates between membranes, the disk membrane cholesterol content can be altered by incubation with cholesterol/phospholipid vesicles. The effect of membrane cholesterol on rhodopsin was investigated using three independent techniques: thermal bleaching, differential scanning calorimetry (DSC) and activation of the cGMP cascade. Rhodopsin exhibited an increased resistance to thermally induced bleaching as the membrane cholesterol level was increased. DSC also indicated that the protein is stabilized by cholesterol in that the Tm increased in response to higher membrane cholesterol. A similar degree of stabilization was observed in both the unbleached and bleached states in the DSC experiments. These results suggest that cholesterol affects the disk membrane properties such that thermally induced unfolding is inhibited, thus stabilizing the rhodopsin structure. Furthermore, high membrane cholesterol inhibited the activation of the cGMP cascade. This is consistent with the stabilization of the metarhodopsin I photointermediate relative to the metarhodopsin II intermediate.