ROLE OF PROTEIN-DISULFIDE INTERCHANGE ENZYME IN LENS

这项研究提案的长期目标是确定 蛋白质-二硫键互换酶(蛋白质-二硫键 异构酶/氧化还原酶)在正常眼晶状体和/或 白内障的形成，特别是在二硫化物的产生上 在白内障中常见的高分子量(HMW)聚集体。这个 酶在这里被称为谷胱甘肽-胰岛素转氢酶(GIT)， 与本实验室以前的报告一致。Git出现了 普遍存在于组织中，包括视网膜；这种酶是否存在于 晶状体或其他眼部组件尚不清楚。在一个人面前 硫醇(如谷胱甘肽、半胱胺)，Git通过二硫键催化 使二硫键的裂解或形成互换 蛋白质(例如，胰岛素、天然或混合的胰岛素原、混合的 核糖核酸酶等)，导致蛋白质失活或激活 底物(包括从一种产品生成聚合产品 蛋白质、胰岛素)。这一试点项目的具体目标是确定 在生命中的任何时候，晶状体中是否含有(1)Git或Git样酶 和/或(2)作为底物(S)起作用的二硫键蛋白(S) Git，导致高分子量聚集体的产生 (HMW)。实验上，对于目标1，获得晶状体(人和/或牛) 不同年龄的人会接受检查，以确定是否有胃肠炎。 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 放射免疫分析)。对于Aim#2，晶状体(从大鼠获得)将是 在器官中短时间至长时间暴露于纯化的git 培养，并监测不透明的形态和HMW 十二烷基硫酸钠-PAGE。这些调查将使我们能够检验这一假设 Git介导的(来自晶状体或视网膜)二硫键交换(较老的)晶状体 蛋白质有助于(或触发)HMW的形成，从而导致 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.