Jensen P E, Stigbrand T
Department of Medical Biochemistry and Biophysics, University of Umeå, Sweden.
Eur J Biochem. 1992 Dec 15;210(3):1071-7. doi: 10.1111/j.1432-1033.1992.tb17513.x.
Different conformational states of human alpha 2-macroglobulin (alpha 2M) and pregnancy zone protein (PZP) were investigated following modifications of the functional sites, i.e. the 'bait' regions and the thiol esters, by use of chymotrypsin, methylamine and dinitrophenylthiocyanate. Gel electrophoresis, mAb (7H11D6 and alpha 1:1) and in vivo plasma clearance were used to describe different molecular states in the proteinase inhibitors. In alpha 2M, in which the thiol ester is broken by binding of methylamine and the 'trap' is closed, cyanylation of the liberated thiol group from the thiol ester modulates reopening of the 'trap' and the 'bait' regions become available for cleavage again. The trapping of proteinases in the cyanylated derivative indicates that the trap functions as in native alpha 2M. In contrast, cyanylation has no effect on proteinase-treated alpha 2M. As demonstrated by binding to mAb, the methylamine and dinitrophenylthiocyanate-treated alpha 2M exposes the receptor-recognition site, but the derivative is not cleared from the circulation in mice. The trap is not functional in PZP. In native PZP and PZP treated with methylamine, the conformational states seem similar. The receptor-recognition sites are not exposed and removal from the circulation in vivo is not seen for these as for the PZP-chymotrypsin complex. Tetramers are only formed when proteinases can be covalently bound to the PZP. Conformational changes are not detected in PZP derivatives in which the thiol ester is treated with methylamine and dinitrophenylthiocyanate. The results suggest that the conformational changes in alpha 2M are generated by mechanisms different to these in PZP. The key structure gearing the conformational changes in alpha 2M is the thiol ester, by which the events 'trapping' and exposure of the receptor-recognition site can be separated. In PZP, the crucial step for the conformational changes is the cleavage of the 'bait' region, since cleavage of the thiol ester does not lead to any detectable conformational changes by the methods used.
通过使用胰凝乳蛋白酶、甲胺和二硝基苯基硫氰酸盐对人α2-巨球蛋白（α2M）和妊娠区蛋白（PZP）的功能位点，即“诱饵”区域和硫酯进行修饰后，研究了它们不同的构象状态。采用凝胶电泳、单克隆抗体（7H11D6和α1:1）以及体内血浆清除率来描述蛋白酶抑制剂中的不同分子状态。在α2M中，硫酯通过甲胺结合而断裂且“陷阱”关闭，硫酯释放的硫醇基团经氰化作用可调节“陷阱”的重新打开，“诱饵”区域再次变得可被切割。蛋白酶在氰化衍生物中的捕获表明该陷阱的功能与天然α2M中的相同。相比之下，氰化对经蛋白酶处理的α2M没有影响。如与单克隆抗体结合所示，经甲胺和二硝基苯基硫氰酸盐处理的α2M暴露了受体识别位点，但该衍生物在小鼠体内并未从循环中清除。该陷阱在PZP中不起作用。在天然PZP和经甲胺处理的PZP中，构象状态似乎相似。受体识别位点未暴露，并且与PZP - 胰凝乳蛋白酶复合物一样，在体内未观察到它们从循环中被清除。只有当蛋白酶能够与PZP共价结合时才会形成四聚体。在用甲胺和二硝基苯基硫氰酸盐处理硫酯的PZP衍生物中未检测到构象变化。结果表明，α2M中的构象变化是由与PZP中不同的机制产生的。驱动α2M构象变化的关键结构是硫酯，通过它可以将“捕获”事件与受体识别位点的暴露分开。在PZP中，构象变化的关键步骤是“诱饵”区域的切割，因为硫酯的切割通过所使用的方法不会导致任何可检测到的构象变化。
