The long-term goal of this project is to investigate the process of oxidative damage o membrane-associated proteins and the ways in which such damage affects tissue junction. Of special interest is the role of nutritional factors; dietary fat, trace elements, (Beta-carotene, and vitamins E and C in promoting or protecting against damage to membranes associated proteins. Oxidative modification of cytosolic and plasma proteins correlates with impaired cellular function in a number of age associated diseases including atherosclerosis, arthritis, muscular dystrophy, cataractogenesis, pulmonary dysfunction and various neurological disorders. Oxidative modification of membrane-associated proteins as been shown to occur and postulated to result in impaired function but the role of the lipid bilayer in mediating such damage is undefined. The role of the lipid bilayer in mediating oxidative damage to membrane associated proteins will be examined in a model system consisting of cytochrome B5 isolated from ovine liver microsomes and incorporated into small unilamellar liposomes. The reconstituted liposomes will be reacted with free radicals generated outside the liposome bilayer by the thermal decomposition of 2,2'-azobis(2-amidinopropane) dihydrochloride. The protein will be studied in liposomes prepared from phosphatidylcholine varying with respect to its susceptibility to lipid peroxidation because of the nature of the esterified fatty acids. Following oxidation, the resulting lipid peroxidation will be correlated with loss of enzymic activity, level of protein oxidation as indicated by protein carbonyl content and protein fragmentation or covalent modification as determined by SDS-PAGE. Specific amino acids which are oxidatively modified will be identified following acid hydrolysis, dabsyl derivatization and reverse phase HPLC analysis with detection at 436 nm. Localization of the modified amino acid residues will be accomplished by appropriate enzymatic proteolysis of the modified protein, separation of the peptides by reverse phase HPLC and then acid hydrolysis and HPLC analysis of the Dabsyl derivatives.
