Prabhakaram M, Ortwerth B J
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Exp Eye Res. 1992 Sep;55(3):451-9. doi: 10.1016/0014-4835(92)90118-c.
Individual lens crystallins were isolated from calf lens extracts and incubated in the presence of ascorbic acid for 3 weeks under aerobic conditions. Both alpha-crystallin and beta H-crystallin rapidly cross-linked to form high molecular weight proteins, which did not enter the resolving gel on SDS-PAGE. Beta L-crystallin was somewhat less reactive, but gamma-crystallin showed little or no crosslinking. Gamma-crystallin, however, was almost equivalent to the other crystallins as a substrate for glycation. This was measured by: (a) the binding of protein to a boronate affinity column; (b) the incorporation of 3H from NaB3H4 into protein; (c) amino acid analysis of the modified proteins to estimate the extent of lysine modification; and (d) the incorporation of [1-14C]ASA into individual crystallins. When the separated crystallins were combined with [125I]gamma-crystallin and incubated with ascorbic acid, radioactivity was readily incorporated into the cross-linked products with other crystallins, but again not with gamma-crystallin itself. Gel filtration chromatography of a mixture of [125I]gamma-crystallin and alpha-crystallin showed the formation of a complex between gamma- and alpha-crystallins. These data suggest that all crystallins are glycated, but that cross-linking occurs preferentially between proteins, which are already bound together non-covalently.
从牛晶状体提取物中分离出单个晶状体蛋白，并在有氧条件下于抗坏血酸存在的情况下孵育3周。α-晶状体蛋白和βH-晶状体蛋白都迅速交联形成高分子量蛋白质，这些蛋白质在SDS-PAGE中不会进入分离胶。βL-晶状体蛋白的反应性稍低，但γ-晶状体蛋白几乎没有交联或交联很少。然而，γ-晶状体蛋白作为糖基化的底物与其他晶状体蛋白几乎相当。这通过以下方法测量：(a)蛋白质与硼酸亲和柱的结合；(b)将NaB3H4中的3H掺入蛋白质；(c)对修饰蛋白质进行氨基酸分析以估计赖氨酸修饰的程度；以及(d)将[1-14C]抗坏血酸掺入单个晶状体蛋白中。当分离的晶状体蛋白与[125I]γ-晶状体蛋白混合并与抗坏血酸一起孵育时，放射性很容易掺入与其他晶状体蛋白交联的产物中，但同样不会掺入γ-晶状体蛋白本身。对[125I]γ-晶状体蛋白和α-晶状体蛋白的混合物进行凝胶过滤色谱分析，结果显示γ-晶状体蛋白和α-晶状体蛋白之间形成了复合物。这些数据表明，所有晶状体蛋白都会发生糖基化，但交联优先发生在已经非共价结合在一起的蛋白质之间。
