CATARACT-RELATED DISULFIDE CROSS-LINKAGES IN CRYSTALLINS

有很多证据表明，白内障，以及正常的 透镜的老化可能随着二硫键交联的形成而发生 在透镜蛋白质中。 这些交联导致形成 大的聚集体，其散射光并导致透镜变成 不透明 拟议调查的两个最重要的目标 是（1）鉴定晶体蛋白中的特定半胱氨酸残基 通过二硫键形成分子间 交联，和（2）鉴定对晶体蛋白的修饰， 在二硫键交联形成之前。 因为透镜 提取物是许多不同晶体蛋白的混合物， 实验将使用两种高度纯化的模型，牛α A2- 和γ II-晶状体蛋白，以表明哪些二硫键交联 在体外形成。 这些模型晶体蛋白将在 与氰酸钾，这是已知的， 白内障的体外和体内研究。 该反应的产物将是 识别和分析，以提高我们的理解， 暴露于氰酸盐，以及其他改变 晶体蛋白的结构，导致形成分子间 透镜蛋白中的交联。 因为有证据表明 阿司匹林抑制白内障的发生， 将鉴定阿司匹林和模型晶体蛋白。 的 生理意义的理解的机制 通过其在体外形成二硫键交联， 通过对分离的透镜蛋白进行类似的分析来确定 来自白内障牛透镜，其中白内障是由 给小牛服用氰酸钾。 拟议 调查将针对获得类似的 白内障患者晶状体的信息。 二硫键高 将分析分子量聚集体以确定 修饰导致这些蛋白质倾向于 形成二硫键交联。 这份详细的调查报告 分子水平上的二硫键是可能的， 基于HPLC和快原子轰击质谱的新分析方法 光谱法将用于鉴定修饰的肽。

There is much evidence indicating that cataract, as well as normal aging of the lens, may occur as disulfide crosslinkages are formed in the lens proteins. These crosslinkages lead to formation of large aggregates, which scatter light and cause the lens to become opaque. The two most important goals of the proposed investigation are to (1) identify specific cysteinyl residues in the crystallins which undergo disulfide bonding to form intermolecular crosslinkages, and (2) identify modifications to crystallins which precede the formation of disulfide crosslinkages. Because lens extracts are mixtures of many different crystallins, initial experiments will use two highly purified models, bovine alpha A2- and gamma II-crystallin, to indicate which disulfide crosslinkages are formed in vitro. These model crystallins will be incubated with potassium cyanate, which is known to cause the formation of cataract in vitro and in vivo. Products of this reaction will be identified and analyzed to improve our understanding of how exposure to cyanate, as well as other substances which change the structure of crystallins, leads to the formation of intermolecular crosslinkages in lens proteins. Since there is some evidence that aspirin inhibits cataractogenisis, the products of the reaction of aspirin and the model crystallins will be identified. The physiological significance of our understanding of the mechanism by which disulfide crosslinkages are formed in vitro will be determined by performing similar analyses on lens proteins isolated from cataractous bovine lens where cataract is induced by the administration of potassium cyanate to a calf. The proposed investigation will then be directed at obtaining similar information from cataractous human lenses. Disulfide-bonded high molecular weight aggregates will be analyzed to determine what modifications have caused a predisposition of these proteins to form disulfide crosslinkages. This detailed investigation of disulfide bonding at the molecular level is possible only because new analytical methods based on HPLC and fast atom bombardment mass spectrometry will be used to identify modified peptides.