LENS PROTEIN GLYCATION & CATARACT DEVELOPMENT

糖尿病患者患白内障的可能性是正常年龄的四到六倍 比正常人群更年轻的年龄。延迟或减少 此类并发症的发生率被认为是由于血糖水平 使之接近正常范围。生命蛋白质的糖基化是一种 高血糖影响组织的方式，如 不依赖于胰岛素的镜片。特定氨基的改性 晶状体蛋白糖基化后的酸性残基导致 蛋白质组织。增加对羟基的暴露 最终也可能发生氧化。这一机制可能会起到 在启动或加强 高分子量(HMW)聚集体形成，蛋白质 溶解不良、混浊和白内障的发展。至 建立晶状体蛋白糖基化与蛋白质之间的关系 白内障的发展我们建议用链脲佐菌素治疗糖尿病 研究大鼠糖基化、高分子的进行性变化 重量(HMW)聚集体的形成和蛋白质的不溶解 在白内障前期和白内障阶段，并研究其影响 醛糖还原酶抑制剂对晶状体蛋白糖基化和HMW的影响 集合体的形成。除了动物研究，长期的 体外糖基化研究将被用来确定这种关系 糖基化和HMW聚集之间。HMW蛋白和 游离晶体蛋白或其他蛋白质的水溶性和尿素- 可溶组分将被分子筛HIGH分离 高效液相色谱分析。特征描述 这些蛋白质将涉及使用反相高效液相色谱进行 蛋白质亚基的分离、氨基酸分析、等电点 聚焦、凝胶电泳法和免疫印迹法。硫醇二硫化物 将利用交换层析法测定 硫醇氧化或二硫化物形成的程度。其影响范围 总的水溶部分和尿素可溶部分的糖基化， 单独的晶体蛋白和聚集体将被确定 通过(~3H)NaBH_4还原、苯基硼酸根- 琼脂糖亲和层析和分子筛层析。这个 将确定每个晶体蛋白亚基的糖基化位点。 糖基化终极产物的存在或因此- 被称为荧光褐变的产品将通过 荧光分光光度计。建议进行的研究包括 预计将证实这一假说：糖基化蛋白 构象变化--硫醇-蛋白质反应性增强 二硫化物-聚集体。

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.