ROLE OF PROTEIN-DISULFIDE INTERCHANGE ENZYME IN LENS

蛋白质-二硫键交换酶在晶状体中的作用

• 批准号: 3426341
• 负责人: DANIEL T ORGANISCIAK
• 金额: $ 2.21万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1987-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3426341
• 关键词: cataract; chemical aggregate; crosslink; disulfide bond; enzyme substrate; gel electrophoresis; human tissue; immunochemistry; lens proteins; organ culture; protein disulfide reductase (glutathione)

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.

本研究计划的长期目标是确定是否 蛋白质二硫键交换酶（蛋白质二硫键 异构酶/氧化还原酶）在正常眼晶状体和/或 白内障的形成，特别是二硫键连接的白内障的形成 白内障中常见的高分子量 (HMW) 聚集体。 这 酶在这里被称为谷胱甘肽-胰岛素转氢酶（GIT）， 为了与该实验室以前的报告保持一致。 胃肠道疾病发生 普遍存在于组织中，包括视网膜；该酶是否存在于 晶状体或其他眼部组件尚不清楚。 在存在一个 硫醇（例如谷胱甘肽、半胱胺），GIT 通过二硫键催化 在某些情况下交换二硫键的裂解或形成 蛋白质（例如胰岛素、天然或炒胰岛素原、炒胰岛素 核糖核酸酶等），导致蛋白质失活或激活 底物（包括从一个物质生成聚合产物） 蛋白质、胰岛素）。 该试点项目的具体目标是确定 生命中任何时候的晶状体是否含有 (1) GIT 或类 GIT 酶 和/或(2)二硫键蛋白质，其作为底物发挥作用 GIT，导​​致高分子量聚集体的生成 （高分子量）。 实验上，对于目标#1，获得了镜片（人类和/或牛） 不同年龄段的人都会接受 GIT 检查，看看是否存在 GIT。 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）。 对于目标#2，镜片（从老鼠身上获得）将是 在器官中短时间或长时间暴露于纯化的胃肠道 培养物，并通过形态学和 HMW 监测不透明度 SDS-PAGE。 这些调查将使我们能够检验以下假设： 胃肠道介导的（来自晶状体或视网膜）（旧）晶状体的二硫键交换 蛋白质有助于（或触发）HMW 的形成，从而促进 某些白内障的发展。