Lipid and Fatty Acid Content of Detergent Resistant Membranes from Bovine Photoreceptor Outer Segments.

Abstract

Abstract: : Purpose: Membrane microdomains contribute to the specificity of cellular function and signaling. One approach to their study is to prepare detergent–resistant lipid rafts. These preparations have been characterized in many cell types, but this is the first characterization of lipid raft phospholipids and fatty acids from photoreceptor outer segments (ROS). Methods: ROS were prepared from light–adapted bovine retinas and solubilized with trituration in a 20gauge needle and 1% Triton X–100 at 4°C. ROS detergent–resistant membranes (DRM) were collected at the 17.1%/20.5% interface after centrifugation in discontinuous sucrose gradients. Relative fraction purity was confirmed by Western blot of the DRM–associated protein, caveolin. Aliquots of ROS and DRM were extracted and lipid classes were separated by 2–dimensional thin–layer chromatography. Fatty acids within each lipid were analyzed by gas chromatography. Results: DRM represented 8% and 3% respectively of total ROS lipid and protein. Relative to ROS, DRM were more enriched in ceramide (CM), sphingomyelin, cholesterol, free fatty acids (FFA), and a form of phosphatidylcholine (PC) that was almost devoid of polyunsaturated fatty acids (PUFA). CM from ROS contained PUFA but no saturated fatty acids while the converse was true of CM from DRM. Similarly, PUFA were more abundant in FFA from ROS than from DRM. Docosahexaenoic acid was diminished in DRM phosphatidylserine, absent in the DRM FFA pool, and equally abundant in ROS and DRM phosphatidylethanolamine. Conclusions: The specialized phospholipid/fatty acid composition of ROS–derived DRM is consistent with findings in other membranes. The possibility of raft–specific, lipid–associated cell signaling is raised by the differences in lipid content of the ROS and DRM. As dietary PUFA content has been shown to influence raft composition in T–cells, there may be effects of diet on ROS DRM.

Keywords: retinal degenerations: cell biology • photoreceptors • lipids