地塞米松与α-晶状体蛋白的结合。

Jobling A I, Stevens A, Augusteyn R C

National Vision Research Institute of Australia, 386 Cardigan Street, Carlton, Victoria 3053, Australia.

Invest Ophthalmol Vis Sci. 2001 Jul;42(8):1829-32.

PURPOSE

Long-term steroid therapy is a known risk factor for the development of posterior subcapsular cataract. Previous work in this laboratory has found soluble lens proteins to bind dexamethasone, but this binding is not due to a glucocorticoid receptor. This study was undertaken to identify the soluble protein or proteins involved in lens glucocorticoid binding.

METHODS

Bovine lens extract was incubated with 5.2 x 10(-)(8) M [(3)H]-dexamethasone for 3 hours, and the distribution of label assessed in the soluble and insoluble fractions after centrifugation. Soluble lens extract was fractionated using gel permeation chromatography to isolate and identify proteins involved in the binding. Total lens proteins, high-molecular-weight proteins, or alpha-crystallin were exposed to dexamethasone and the protein bound steroid measured after separation of free and bound ligand on a gel chromatography column. Scatchard analysis was used to determine dexamethasone-binding parameters. Sequence comparisons between bovine alphaA- and alphaB-crystallins and glucocorticoid-binding proteins were performed using a sequence-alignment program.

RESULTS

Of the total dexamethasone bound in lens extract, soluble proteins were found to account for 52%. The majority of the soluble protein-bound dexamethasone coeluted with the high-molecular-weight proteins that consisted mainly of alpha-crystallin. Binding studies with isolated proteins showed that alpha-crystallin accounted for more than 98% of total soluble dexamethasone binding in the lens. Scatchard analysis of steroid binding showed it to be a nonspecific partitioning event. Sequence comparisons between alphaA- and alphaB-crystallins and various glucocorticoid-binding proteins showed the lens proteins to have three regions of sequence homology with yeast corticosteroid-binding protein.

CONCLUSIONS

alpha-Crystallin is the principal soluble glucocorticoid binding protein in the lens. The steroid association is described by nonspecific partitioning and may be related to the unique structural characteristics of the protein. The nonspecific association with alpha-crystallin is not thought to be functional; however, it may aid in the increased covalent steroid modification observed for this protein.

目的

长期使用类固醇疗法是后囊下白内障发生的已知风险因素。本实验室先前的研究发现可溶性晶状体蛋白可结合地塞米松，但这种结合并非由糖皮质激素受体介导。本研究旨在鉴定参与晶状体糖皮质激素结合的可溶性蛋白。

方法

将牛晶状体提取物与5.2×10⁻⁸ M [³H] -地塞米松孵育3小时，离心后评估可溶性和不可溶性部分中标记物的分布。使用凝胶渗透色谱法对可溶性晶状体提取物进行分级分离，以分离和鉴定参与结合的蛋白质。将总晶状体蛋白、高分子量蛋白或α-晶状体蛋白暴露于地塞米松，在凝胶色谱柱上分离游离和结合配体后，测量结合类固醇的蛋白质。使用Scatchard分析来确定地塞米松结合参数。使用序列比对程序对牛αA-和αB-晶状体蛋白与糖皮质激素结合蛋白进行序列比较。

结果

在晶状体提取物中结合的总地塞米松中，发现可溶性蛋白占52%。大多数与可溶性蛋白结合的地塞米松与主要由α-晶状体蛋白组成的高分子量蛋白共洗脱。对分离蛋白的结合研究表明，α-晶状体蛋白占晶状体中可溶性地塞米松总结合量的98%以上。类固醇结合的Scatchard分析表明这是一个非特异性分配事件。αA-和αB-晶状体蛋白与各种糖皮质激素结合蛋白的序列比较表明，晶状体蛋白与酵母皮质类固醇结合蛋白有三个序列同源区域。