Ortwerth B J, Sharma K K, Olesen P R
Mason Institute of Ophthalmology, Columbia, MO.
Exp Eye Res. 1992 Apr;54(4):573-81. doi: 10.1016/0014-4835(92)90136-g.
Preparations of alpha-crystallin from bovine and human lens exhibited elastase inhibitor activity with a specific activity of 100-250 U mg-1 protein. A washed water-insoluble fraction from bovine, human and cataractous lens nuclei, when solubilized by sonication, gave specific activities of 910, 950 and 1270 U mg-1, respectively. Disaggregation of these water-insoluble fractions in 8.0 M urea, with subsequent reaggregation by urea removal, resulted in a decrease in inhibitor activity. Agarose A-5m gel filtration chromatography after the urea treatment resolved a residual high molecular weight (HMW) fraction and a peak which eluted at the position of water soluble alpha-crystallin. Assays showed that the urea-induced 'alpha-crystallin' peaks from all three preparations had specific activities, equivalent to native alpha-crystallin, whereas the HMW fractions retained their original high specific activity. We conclude that the increased elastase inhibitor activity of the water-insoluble fraction is a property of the aggregate state of the component alpha-crystallin molecules, which is lost upon reaggregation to an 800-kDa alpha-crystallin. Amino acid analysis of the bovine water-insoluble fraction suggested a content of 85-90% alpha-crystallin and 10-15% beta H-crystallin, which was confirmed by SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)
从牛和人晶状体中制备的α-晶状体蛋白制剂表现出弹性蛋白酶抑制活性，比活性为100 - 250 U mg-1蛋白。牛、人及白内障晶状体核的水洗水不溶性部分，经超声溶解后，比活性分别为910、950和1270 U mg-1。这些水不溶性部分在8.0 M尿素中解聚，随后通过去除尿素进行再聚集，导致抑制活性降低。尿素处理后的琼脂糖A - 5m凝胶过滤色谱分离出一个残留的高分子量（HMW）部分和一个在水溶性α-晶状体蛋白位置洗脱的峰。分析表明，所有三种制剂中尿素诱导的“α-晶状体蛋白”峰具有与天然α-晶状体蛋白相当的比活性，而HMW部分保留了其原始的高比活性。我们得出结论，水不溶性部分弹性蛋白酶抑制活性的增加是组成α-晶状体蛋白分子聚集状态的一种特性，在重新聚集为800 kDa的α-晶状体蛋白时会丧失。对牛水不溶性部分的氨基酸分析表明，其α-晶状体蛋白含量为85 - 90%，βH-晶状体蛋白含量为10 - 15%，这通过SDS-PAGE得到了证实。（摘要截短至250字）
