Gerbod M C, Janciauskiene S, Jeppsson J O, Eriksson S
Department of Medicine, Lund University, Malmö, S-20502, Sweden.
Arch Biochem Biophys. 1998 Mar 15;351(2):167-74. doi: 10.1006/abbi.1997.0541.
We describe here an in vitro effect of lithocholic acid (LA), a secondary, hydrophobic bile acid, on the rate of polymerization of mutant, Z and wild-type, M alpha-1-antitrypsin (AAT). Using thioflavine T fluorescence and turbidity assays we demonstrated that the rate of aggregation for the Z AAT in the presence of LA at a molar ratio of 1:5 AAT to LA, in Tris-buffered saline, pH 7.4, is at least twice that of the Z protein alone or the M variant with and without LA. Also, Z AAT incubated for 48 h at room temperature had more than 50% diminished antielastase activity, while M AAT had only a 25% reduction in activity. Analysis of the AAT and AAT-LA samples after cleavage with pancreatic elastase by SDS-PAGE 10% gels showed that interaction between Z or M AAT and LA abolishes their ability to form SDS stable complexes with an enzyme and both of these forms of AAT showed elastase substrate behavior. Furthermore, Z as well as M AAT incubated with LA at 41 degrees C and cleaved with elastase showed only 80 to 60% increased thermal stability compared to 100% stabilization for the cleaved AAT alone in the absence of LA. These observations suggest that a rearrangement of the AAT molecule as a result of interactions with LA increases aggregation of AAT and diminishes its inhibitory activity.
我们在此描述了石胆酸（LA）（一种二级疏水胆汁酸）对突变型Z和野生型Mα-1抗胰蛋白酶（AAT）聚合速率的体外影响。使用硫黄素T荧光和浊度测定法，我们证明，在pH 7.4的Tris缓冲盐溶液中，当AAT与LA的摩尔比为1:5时，Z AAT在LA存在下的聚集速率至少是单独的Z蛋白或有无LA存在时M变体的两倍。此外，Z AAT在室温下孵育48小时后，其抗弹性蛋白酶活性降低了50%以上，而M AAT的活性仅降低了25%。用10%的SDS-PAGE凝胶对经胰弹性蛋白酶切割后的AAT和AAT-LA样品进行分析表明，Z或M AAT与LA之间的相互作用消除了它们与该酶形成SDS稳定复合物的能力，并且这两种形式的AAT均表现出弹性蛋白酶底物行为。此外，与在无LA情况下单独切割的AAT 100%的热稳定性相比，在41℃下与LA一起孵育并经弹性蛋白酶切割的Z以及M AAT仅表现出80%至60%的热稳定性增加。这些观察结果表明，由于与LA相互作用导致的AAT分子重排增加了AAT的聚集并降低了其抑制活性。
