SPECTROSCOPIC & RELATED STUDIES ON LENS MEMBRANE LIPIDS

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The overall objective of this proposal is to study the regional conformation, composition and structural characteristics of lens membrane lipids and proteins, and relate membrane composition to membrane structure and performance (i.e. permeability, cation pump activity). We will use state of the art spectroscopic techniques to characterize normal rabbit and human lens membranes and to compare them to cataractous human lens membranes and membranes subjected to a variety of physiochemical stresses. Our hypothesis is that alterations in membrane lipid composition and structure influence membrane performance. Raman and infrared spectroscopies are among the tools that will be used to pursue six aims to test our single hypothesis. Tissues: Human lenses will be studied with respect to lens-regional changes with age and cataract type. l. What is the lipid headgroup composition in the tissues above and what is the primary structure of the unknown lipid that comprises 60% of the phospholipids? 2. What is the degree of lipid oxidation in the tissues above? oxidation will be assessed by measuring: lipid -OH and -OOH, cholesterol modifications, green and blue fluorophores and saturation. 3. How do intrinsic and extrinsic proteins influence lipid-lipid and lipid-protein interactions and, are there secondary and tertiary structural differences in membrane proteins in the tissues above? 4. What is the impact of: oxidative stress, calcium and cholesterol/filipin upon the spectroscopic characteristics of lens cell membrane and membrane lipids? 5. Do secondary products of lipid oxidation alter the secondary and tertiary structure of purified bovine crystallins or human lens homogenates? 6. What is the correlation of membrane lipid composition and structure with lens membrane performance, i.e Ca-ATPase, Na, K-ATPase and MIP26 function?