摘要
目的 :探讨糖基化对α 晶状体蛋白的修饰 .方法 :分离牛αL 和 βL 晶状体蛋白 .分别与 1 0 0mmol/L 6 磷酸果糖(F6P)和核糖孵育 0～ 2 5d,于 0 ,1 5和 2 5d监测 2 0 0～ 6 0 0nm波长范围的吸光度 (A)值 ,色氨酸和非色氨酸荧光值 ,SDS 聚丙烯酰胺凝胶电泳分析蛋白质交联物 .测定αL 晶状体蛋白对加热诱导βL 晶状体蛋白凝聚的保护作为分子伴侣活性 .结果 :F6P和核糖对αL 晶状体蛋白的修饰 ,主要表现为时间依赖性的色氨酸荧光强度降低 ,非色氨酸荧光强度增强 ,交联物形成 ,分子伴侣活性降低 ,F6P较核糖的修饰作用显著 .孵育 4d ,F6P组伴侣活性下降约 2 4 % (P <0 .0 1 ) ,1 5d ,伴侣活性几乎殆尽 ,而核糖组伴侣活性下降约 6 0 % (P <0 .0 1 ) .结论 :糖基化可导致α 晶状体蛋白色氨酸残基内环境的构象改变 ,形成交联物 。
AIM: To investigate the effects of modification on α crystallin by glycation. METHODS: The α L and β L crystallin were separated from bovine lenses on Sephacryl S 300HR and then were incubated with 100 mmol/L fructose 6 phosphate (F6P) and 100 mmol/L ribose at 37℃, respectively. The absorbance at 200-600 nm, the tryptophan fluorescence and non tryptophan fluorescence were observed after 0, 15 and 25 d of incubation. The extent of crosslinking was detected by SDS PAGE. The chaperone activity of α crystallin was assayed by measuring heat induced aggregation and scattering of β L crystallin. RESULTS: The major modification of α L crystallin by F6P and ribose showed that the fluorescence intensity of the tryptophan decreased and the non tryptophan increased in a time dependent manner. SDS PAGE results showed that glycation led to crosslinking formation of α L crystallin. F6P presented more effective cross linked α L crystallin than ribose. The protective effects of α L crystallin against thermal aggregation β L crystallin decreased. After 4 d of incubation, the protective ability in α L crystallin incubated with F6P diminished approximately 24% ( P <0.01). At 15 days, the chaperone activity decreased nearly 100% in F6P incubation, whereas roughly 60% in ribose incubation ( P <0.01). CONCLUSION: Glycation may induce a conformational change within the environment of a tryptophan residue of the modified α crystallin and form the crosslinking, thus leading to the reduction of its chaperone activity.
出处
《第四军医大学学报》
北大核心
2004年第11期994-997,共4页
Journal of the Fourth Military Medical University
