ROLE OF PROTEIN-DISULFIDE INTERCHANGE ENZYME IN LENS

这项研究计划的长期目标是确定 蛋白质-二硫键交换酶（蛋白质-二硫键 异构酶/氧化还原酶）在正常的眼睛透镜和/或在 白内障形成，特别是在二硫键连接的白内障形成中， 高分子量（HMW）聚集体常见于白内障。 的 酶在此称为谷胱甘肽-胰岛素转氢酶（GIT）， 与本实验室以前的报告一致。 GIT发生 普遍存在于组织中，包括视网膜;该酶是否存在于 透镜或其它目镜部件是未知的。 存在下 硫醇（例如，谷胱甘肽，半胱胺），GIT通过二硫化物催化 在某些情况下交换二硫键的断裂或形成 蛋白质（例如，天然胰岛素或混杂胰岛素原，混杂 核糖核酸酶等），导致蛋白质的失活或活化 基质（包括从一个基质生成聚集产物） 蛋白质、胰岛素）。 该试点项目的具体目标是确定 生命中任何时候的透镜是否含有（1）GIT或GIT样酶 和/或（2）二硫键-蛋白，其作为底物， GIT，导致产生高分子量聚集体 （HMW）。 在实验上，对于目标#1，获得了晶状体（人和/或牛） 在不同的年龄将检查GIT的存在， 酶学和免疫学方法（免疫扩散试验， 定量抗原-抗体滴定、免疫印迹和 放射免疫测定法）。 对于目标#2，将使用透镜（从大鼠中获得） 在器官中短期至长期暴露于纯化GIT 培养，并通过以下方法监测形态学不透明度和HMW SDS-PAGE。 这些调查将使我们能够检验假设， GIT介导的（来自透镜或视网膜）（老年）透镜的二硫键交换 蛋白质有助于（或触发）HMW的形成， 某些白内障的发展。

The long-range goal of this research proposal is to determine whether the protein-disulfide interchange enzyme (protein-disulfide isomerase/oxidoreductase) has any function in the normal eye lens and/or in cataract formation, in particular in the generation of disulfide-linked high molecular weight (HMW) aggregates commonly seen in cataracts. The enzyme is referred to here as glutathione-insulin transhydrogenase (GIT), for consistency with previous reports form this laboratory. GIT occurs ubiquitously in tissues, including retina; whether the enzyme is present in the lens or in other ocular components is not known. In the presence of a thiol (e.g., glutathione, cysteamine), GIT catalyzes via disulfide interchange the cleavage or formation of disulfide bonds in certain proteins (e.g., insulin, native or scrambled proinsulin, scrambled ribonuclease, etc.), resulting in inactivation or activation of the protein substrate (including the generation of an aggregated product from one protein, insulin). Specific aims of this pilot project are to determine whether the lens at any time in life contains (1) GIT or GIT-like enzyme and/or (2) a disulfide-protein(s) which funcitons as a substrate(s) for GIT, resulting in the generation of high molecular weight aggregates (HMW). Experimentally, for aim #1, lenses (human and/or bovine) obtained at different ages will be examined for the presence of GIT both by enzymological and immunological methods (immunodiffusion tests, quantitative antigen-antibody titrations, immunoblots and radioimmunoassay). For aim #2, the lens (obtained from rats) will be exposed to purified GIT over short to prolonged periods of time in organ culture, and monitored for opacity morphologically and for HMW by SDS-PAGE. These investigations will allow us to test the hypothesis that GIT-mediated (from lens or retina) disulfide interchange of (older) lens proteins contributes to (or triggers) the formation of HMW and thus to the development of certain cataracts.