Moreau T, Gutman N, Faucher D, Gauthier F
Centre National de la Recherche Scientifique, U.R.A. 334, University François Rabelais, Tours, France.
J Biol Chem. 1989 Mar 15;264(8):4298-303.
Limited proteolysis of T-kininogen by heterologous and homologous endopeptidases (bovine trypsin, human leukocyte elastase, rat submaxillary gland endopeptidase k, and rat mast cell chymase) produced similar fragmentation. Amino-terminal sequence analysis of whole T-kininogen lysates and purified proteolytic fragments identified four susceptible regions which contained all the preferential cleavage sites for these proteinases. Two of these susceptible regions were close to the junction between heavy chain cystatin-like domains, the third was in the kinin-containing region, and the fourth was close to the carboxyl terminus of the T-kininogen light chain. There was only one primary site for each proteinase in the kinin-containing region, which explains why catalytic amounts of these proteinases did not release immunoreactive kinin from this kininogen. However, preferential cleavage of T-kininogen close to the junction between cystatin-like domains released fragments which, provided they included cystatin-like domains 2 and/or 3, strongly inhibited papain and cathepsin L. The fragments were inhibitory even when parts of the amino-terminal ends of the domains were lacking. The highly conserved glycyl residue, thought to be involved in the inhibitory reactive site of cystatin-like inhibitors, was not required in purified domain 3 for inhibition of cathepsin L.
用异源和同源内肽酶（牛胰蛋白酶、人白细胞弹性蛋白酶、大鼠颌下腺内肽酶k和大鼠肥大细胞糜酶）对T-激肽原进行有限的蛋白水解，产生了相似的片段化。对整个T-激肽原裂解物和纯化的蛋白水解片段进行氨基末端序列分析，确定了四个敏感区域，这些区域包含了这些蛋白酶的所有优先切割位点。其中两个敏感区域靠近重链胱抑素样结构域之间的连接处，第三个在含激肽区域，第四个靠近T-激肽原轻链的羧基末端。在含激肽区域，每种蛋白酶只有一个主要切割位点，这就解释了为什么这些蛋白酶的催化量不能从这种激肽原中释放出免疫反应性激肽。然而，在胱抑素样结构域之间的连接处附近对T-激肽原进行优先切割会释放出片段，只要这些片段包含胱抑素样结构域2和/或3，就会强烈抑制木瓜蛋白酶和组织蛋白酶L。即使这些结构域的氨基末端部分缺失，这些片段仍具有抑制作用。纯化的结构域3抑制组织蛋白酶L时，并不需要被认为参与胱抑素样抑制剂抑制活性位点的高度保守的甘氨酰残基。
