LENS PROTEIN GLYCATION & CATARACT DEVELOPMENT

糖尿病患者患白内障的可能性是普通人的四到六倍， 比正常人群更年轻。 延迟或减少 这些并发症的发生率被视为葡萄糖水平， 使其达到正常范围。 重要蛋白质的糖基化是一种 葡萄糖升高可影响组织的方式， 不依赖胰岛素的透镜。 特定氨基的修饰 糖基化导致晶体蛋白的酸性残基破坏， 蛋白质组织 巯基暴露增加， 也可能发生最终的氧化。 这一机制可以发挥 无论是在启动或在增强的重要作用， 高分子量（HMW）聚集体形成，蛋白质 不溶解、混浊和白内障发展。 到 建立晶体蛋白糖基化与 白内障的发展，我们建议使用链脲佐菌素糖尿病 研究大鼠糖基化、高分子 重量（HMW）聚集体形成和蛋白质不溶 在白内障前期和白内障阶段， 醛糖还原酶抑制剂对透镜蛋白糖基化和HMW的影响 聚集体形成 除了动物研究，长期在 将利用体外糖化研究来确定 糖基化和HMW聚集之间的关系。 HMW蛋白和 游离晶状体蛋白或其他水溶性蛋白质和尿素， 可溶性馏分将通过分子筛分离， 高效液相色谱法（HPLC）。 表征 蛋白质将涉及利用反相HPLC， 蛋白质亚基分离，氨基酸分析，等电 聚焦、凝胶电泳和免疫印迹。 硫醇二硫化物 交换色谱法将用于确定 硫醇氧化或二硫化物形成的程度。 的程度 总水溶性和尿素溶性部分的糖化， 将测定单个晶体蛋白和聚集体 通过（3 H）NaBH 4还原、苯基硼氢化物- 琼脂糖亲和层析和分子筛HPLC。 的 将鉴定每个晶状体蛋白亚基的糖化位点。 糖基化最终产物的存在或如此- 称为荧光布朗宁产品将被监测与 荧光分光光度计 拟议的研究是 预计将证实假设：糖基化蛋白 构象变化-增加巯基-蛋白质的反应性 二硫化物聚集。

Diabetics are four to six times more likely to develop cataracts at a younger age than the normal population. Delayed or decreased incidence of such complications are seen as glucose levels are brought toward normal range. Glycation of vital proteins is one of the means by which elevated glucose can affect tissues such as lens that are insulin independent. Modification of specific amino acid residues of crystallins by glycation leads to disruption of protein organization. Increased exposure of sulfhydryl groups for eventual oxidation also may occur. This mechanism could play a significant role either in the initiation or in the enhancement of high molecular weight (HMW) aggregate formation, protein insolubilization, opacification, and cataract development. To establish the relationship between crystallin glycation and cataract development we propose to use streptozotocin diabetic rats to study the progressive changes in glycation, high molecular weight (HMW) aggregate formation, and protein insolubilization during precataract and cataract stages and to study the influence of aldose reductase inhibitors on lens protein glycation and HMW aggregate formation. In addition to animal studies, long-term in vitro glycation studies will be utilized to establish the relationship between glycation and HMW aggregation. The HMW proteins and free crystallins or other proteins of the water-soluble and urea- soluble fractions will be separated by molecular sieve high performance liquid chromatography (HPLC). Characterization of the proteins will involve utilization of reverse-phase HPLC for separation of protein subunits, amino acid analysis, isoelectric focusing, gel electrophoresis and immunoblotting. Thiol-disulfide exchange chromatography will be utilized for determining the extent of thiol oxidation or disulfide formation. The extent of glycation of the total water-soluble and urea-soluble fractions, the individual crystallins, and the aggregates will be determined by a combination of (3H)NaBH4 reduction, phenylboranate- agarose affinity chromatography and molecular sieve HPLC. The sites of glycation of each crystallin subunit will be identified. The presence of the ultimate products of glycation or the so- called fluorescent browning products will be monitored with a flourescence spectrophotometer. The proposed studies are expected to confirm the hypothesis: glycation-protein conformational change-increased reactivity of thiols-protein disulfides-aggregation.