PROTEINS OF NORMAL AND CATARACTOUS LENSES

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Our objective is to determine the relationship of oxidation to the formation of human senile cataract and to define conditions in the lens which may promote oxidation. Emphasis will be placed on investigating the oxidation of lens proteins, particularly the amino acid residues of cysteine, methionine and tyrosine. Oxidized lens proteins similar to those which have been found to be present in human senile cataract will be produced in animal lenses by methods involving use of X-irradiation, H2O2 and hyperbaric oxygen. Possible mechanisms for the crosslinking of lens crystallins with each other and with membrane proteins will be examined and the crystallins which form disulfide crosslinks with membranes in X-ray cataract will be identified. Clues to the mechanism of formation of human nuclear cataract may be obtained from studies with hyperbaric oxygen both in vivo and in vitro. Diverse techniques of chromatography, electrophoresis, amino acid analysis, spectroscopy, immunochemistry and electron microscopy will be employed. Silver staining techniques will be used to investigate the leakage of crystallins from X-rayed lenses into aqueous humor. Peptide mapping will be carried out to compare the lens membrane polypeptides MP22 and MP26 in X-ray cataract. Investigations of the role of sulfhydryl groups in lens membranes of human and animal lenses will be aided by the use of fluorescent and radioactive sulfhydryl probes in conjunction with SDA-PAGE. The role of the glutathione redox cycle in defending the lens against oxidative damage will be examined in detail, particularly in specific regions of the lens. The possible involvement of GSH in the repair of oxidized lens membranes and restoration of normal membrane permeability will be studied in cultured lenses following treatment with an inhibitor of glutathione reductase and H2O2. Radioactive glycine and glutamic acid will be used to measure the turnover rate of GSH in cultured lens epithelial cells. Methods will be employed to isolate the anterior and posterior surfaces of the lens in order to investigate the detoxification of H2O2 in the two regions. The effect of aging on the ability of the lens to detoxify H2O2 will be examined in human lenses and in cultured rabbit lens epithelial cells.