CATARACT-RELATED DISULFIDE CROSS-LINKAGES IN CRYSTALLINS

有许多证据表明，白内障和正常的 当形成二硫键交联剂时，可能会发生晶状体老化 在晶状体蛋白质中。这些交叉连接导致了 大团聚体，它散射光线并导致晶状体变成 不透明。拟议调查的两个最重要的目标 是为了(1)鉴定晶体蛋白中特定的半胱氨酸基残基 它们通过二硫键形成分子间 交联键，以及(2)确定对晶体蛋白的修饰 在形成二硫键交联键之前。因为镜头 提取物是许多不同晶体蛋白的混合物，最初 实验将使用两个高度纯化的模型，牛αA2- 和Gamma II-晶状体蛋白，以指示哪些二硫键交联键 是在体外形成的。这些模型晶体蛋白将被孵化 氰酸钾，这是已知的导致形成 体外和体内的白内障。该反应的产物将是 识别和分析，以提高我们对 暴露在氰酸盐以及其他会改变人体健康的物质中 晶体蛋白的结构，导致分子间的形成 晶状体蛋白中的交联键。因为有一些证据表明 阿司匹林抑制白内障形成，这是阿司匹林 阿司匹林和模型晶体蛋白将被识别。这个 我们理解这一机制的生理学意义 在体外形成二硫键的方法将是 通过对分离的晶状体蛋白进行类似的分析而确定 来自白内障牛晶状体，其中白内障是由 给小牛注射氰酸钾。建议数 然后将进行调查，以获得类似的 来自患有白内障的人类晶状体的信息。二硫键高 将分析分子量聚集体以确定 修饰已经导致这些蛋白质容易发生 形成二硫键交联剂。这项详细的调查 分子水平上的二硫键之所以可能，只是因为 基于高效液相色谱和快原子轰击质量的分析新方法 光谱分析将用于鉴定修饰的多肽。

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.