REINVESTIGATION OF THE FATTY-ACID CONTENT OF BOVINE, RAT AND FROG RETINAL ROD OUTER SEGMENTS

Continuous-sucrose gradient centrifugation of whole retina homogenates from bovine, frog and rat were found to yield two major bands of rhodopsin-containing material having distinctly different polyunsaturated fatty acid (PUFA) contents. The lowest density band has the highest purity rod outer segment (ROS) membranes and the highest PUFA content whereas the higher density band has a lower PUFA content and contains non-ROS membrane impurities. It follows, that in ROS preparations from whole retinal homogenates in which these two bands are pooled, the measured PUFA content will be lower than that of the purest ROS band. We find that the highest purity ROS band from bovine, frog and rat retinal homogenates have weight-percents of docosahexaenoic acid (22:6ω3) of 50·7±1·4, 50·9±0·6, and 46·2±3·6, respectively, which is equivalent to 47·1, 46·9, and 43·3 mol-percent. Two ROS bands were also isolated from homogenates of semipurified ROS removed from retinas by shaking (i.e. the shakate preparation). Both shakate bands have similar PUFA contents (with weight-percent 22:6ω3 of ≈51·5) and similar phospholipid: rhodopsin ratios. The more dense band has a significantly higher 280 nm/Δ500 nm ratio which probably reflects some nonrhodopsin protein retained in the ROS by the intact plasma membrane. The lowest density band has ROS with disrupted plasma membranes whereas the more dense band has intact plasma membranes. Homogenization of whole retinas introduces considerable non-ROS membrane contamination into the more dense ROS band. We have developed specific procedures (i.e. use of 0·1 mm CaEDTA and an inert argon atmosphere) that inhibit ROS lipid autoxidation and loss of PUFA. The results indicate that if adequate levels of vitamin E are present the maintenance of antioxidant conditions during preparation procedure is not a highly critical factor in determining ROS PUFA content. © 1979.