ASPARTIC ACID RACEMIZATION IN RELATION TO HUMAN CATARACTOGENESIS

老年公民正在迅速成为美国人口中更大的比例。随着这一趋势的发展，这部分人口中对功能残疾的关注日益增加。视力障碍，如白内障和黄斑变性，是导致失明的主要原因，并构成老年人功能障碍的主要部分。老年性白内障至少部分是由于老化过程中晶状体蛋白质的化学修饰。氨基酸外消旋是已报道的与年龄相关的变化之一。它涉及蛋白质氨基酸的L形式到其自然不常见的D形式的立体反转。这种构象变化可能导致结构变化，最终可能改变修饰蛋白的功能(S)。过去的研究已经记录了老年白内障人晶状体中特定天冬氨酸残基的D/L比值增加，并表明外消旋化可能参与年龄相关性白内障的发病机制，这将有助于制定治疗策略，或至少推迟这种致残性疾病的发生。该项目的长期目标是确定人眼晶状体蛋白中天冬氨酸外消旋的程度是否与年龄相关性白内障的发生率相关。具体目的是利用更可靠的敏化方法，定量比较从白内障和年龄匹配的正常人晶状体中分离出的α-A晶体中Asp-58和Asp-151以及α-B晶体蛋白中Asp-36和Asp-62的消旋作用。这将通过用马菲试剂衍生化的多肽片段(包含指定的天冬氨酸残基)的高效液相分析来完成。天冬氨酸外消旋也将在来自白内障和年龄匹配的正常晶状体的水不溶、尿素可溶的蛋白质组分中进行定量，使用一种最小化背景外消旋的方法，并通过D-天冬氨酸氧化酶的氧化来估计D-天冬氨酸。

Older citizens are fast becoming a greater percentage of the American population. With this trend, there is increasing concern over functional disabilities amongst this segment of the population. Visual disorders like cataract and macular degeneration, are the leading causes of blindness and constitute a major part of functional disabilities amongst older-persons. Age-related cataract is due, at least in part, to chemical modifications in lens proteins during the aging process. Amino acid racemization is one of the age-related changes that have been reported. It involves the stereoinversion of the L-form of a protein amino-acid, to its naturally uncommon D-form. This conformational change may lead to a structural change that may ultimately alter the function(s) of the modified protein. Past studies have documented increased D/L rations of specific aspartate residues of alpha-A crystallins from aged, cataractous human lenses and suggested a possible involvement of racemization in the pathogenesis of age-related cataract will aid in developing strategies for treatment, or at least, delaying the onset of this disabling disease. The long term objective of this project is to determine if the extent of aspartic acid racemization in human eye lens proteins, correlates with incidence of age-related cataract. The specific objectives are to utilize more reliable, sensitize methods, to quantitatively compare racemization of Asp-58, Asp-151 in alpha-A crystalline, and Asp-36, Asp-62 of alpha- B crystallins, isolated from cataractous and age-matched normal human lenses. This will be done by HPLC analysis of peptide fragments (containing the specified aspartate residues), derivatized with Marfey's reagent. Aspartic acid racemization will also be quantitated in water insoluble, urea-soluble protein fractions from cataractous and age- matched normal lenses, using a method that minimizes background racemization and estimates D-aspartic acid by oxidation with D-aspartate oxidase.