PROTEINS OF NORMAL AND CATARACTOUS LENSES

我们的目标是确定氧化与 人老年性白内障的形成并确定透镜中的条件 其可促进氧化。 重点将放在调查 透镜蛋白的氧化，特别是 半胱氨酸、甲硫氨酸和酪氨酸。 氧化的透镜蛋白与那些类似 已经发现存在于人类老年性白内障中的蛋白质， 通过使用X射线照射、H2 O2 和高压氧 透镜交联的可能机制 将检查晶体蛋白彼此之间以及与膜蛋白之间的相互作用， 在X射线照射下与细胞膜形成二硫键交联的晶体蛋白 白内障将被识别。 人类基因组形成机制的线索 核性白内障可以从高压氧的研究中获得， 在体内和体外。 色谱、电泳、氨基酸分析等多种技术， 将使用光谱学、免疫化学和电子显微镜。 将使用银染色技术研究 晶体蛋白从X光晶状体进入房水。 肽图谱将 进行比较透镜膜多肽MP22和MP26， X光白内障 透镜中巯基作用的研究 人类和动物晶状体的膜将通过使用 荧光和放射性巯基探针结合SDA-PAGE。 谷胱甘肽氧化还原循环在保护透镜免受 氧化损伤将详细检查，特别是在特定的 透镜的区域。 谷胱甘肽可能参与修复 氧化的透镜膜和正常膜渗透性的恢复 将在培养的晶状体中进行研究， 谷胱甘肽还原酶和H2 O2。 放射性甘氨酸和谷氨酸 用于测定培养的透镜上皮细胞中GSH的周转率 细胞 将采用方法分离前、后 透镜的表面，以研究H2 O2在 这两个地区。 老化对透镜 将在人晶状体和培养的兔透镜中检测解毒H2 O2 上皮细胞

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.