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.
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