Sharma K K, Olesen P R, Ortwerth B J
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Biochim Biophys Acta. 1987 Sep 24;915(2):284-91. doi: 10.1016/0167-4838(87)90311-6.
One of the major lens-structural proteins, alpha-crystallin, is a multimeric protein containing 40 subunits of approx. 20 kDa each. There are two subunit types with distinct but similar structures. This protein was capable of inhibiting trypsin, chymotrypsin and elastase, but had no effect on thrombin or kallikrein. Complete inhibition was not observed, but rather plateau levels of inhibition were obtained in each case. Maximum inhibition was observed at a ratio of 1 mol of alpha-crystallin for every 9-10 mol of trypsin. alpha-Crystallin also inhibited the labeling of the active site of trypsin by [3H]diisopropyl fluorophosphate (DFP). Greater than 90% inhibition of DFP labeling was observed at a ratio of 1 mol of alpha-crystallin for every 7-8 mol of trypsin. Both trypsin and [3H]DFP-labeled trypsin formed a complex with alpha-crystallin, as demonstrated by gel-filtration chromatography. The active site of trypsin when bound to alpha-crystallin was still capable of reacting with p-nitrophenyl p-guanidobenzoate and soybean trypsin inhibitor, but was inaccessible to alpha 1-antitrypsin. These data suggest that alpha-crystallin acts as a multivalent modified inhibitor which is consistent with the proposed quaternary structure of alpha-crystallin.
主要晶状体结构蛋白之一α-晶体蛋白是一种多聚体蛋白，由约40个亚基组成，每个亚基的分子量约为20 kDa。有两种亚基类型，其结构不同但相似。这种蛋白能够抑制胰蛋白酶、胰凝乳蛋白酶和弹性蛋白酶，但对凝血酶或激肽释放酶没有作用。未观察到完全抑制，而是在每种情况下都获得了抑制的平稳水平。在α-晶体蛋白与胰蛋白酶的摩尔比为1:9 - 10时观察到最大抑制作用。α-晶体蛋白还抑制了[3H]二异丙基氟磷酸酯（DFP）对胰蛋白酶活性位点的标记。在α-晶体蛋白与胰蛋白酶的摩尔比为1:7 - 8时，观察到对DFP标记的抑制率大于90%。凝胶过滤色谱法证明，胰蛋白酶和[3H]DFP标记的胰蛋白酶都与α-晶体蛋白形成了复合物。与α-晶体蛋白结合时，胰蛋白酶的活性位点仍能与对硝基苯基对胍基苯甲酸酯和大豆胰蛋白酶抑制剂反应，但对α1-抗胰蛋白酶不可接近。这些数据表明，α-晶体蛋白作为一种多价修饰抑制剂，这与所提出的α-晶体蛋白的四级结构是一致的。
