Itoh Y, Binner S, Nagase H
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160-7421, USA.
Biochem J. 1995 Jun 1;308 ( Pt 2)(Pt 2):645-51. doi: 10.1042/bj3080645.
Tissue inhibitor of metalloproteinases (TIMP)-2 forms a noncovalent complex with the precursor of matrix metalloproteinase 2 (proMMP-2, progelatinase A) through interaction of the C-terminal domain of each molecule. We have isolated the proMMP-2-TIMP-2 complex from the medium of human uterine cervical fibroblasts and investigated the processes involved in its activation by 4-aminophenylmercuric acetate (APMA). The treatment of the complex with APMA-activated proMMP-2 by disrupting the Cys73-Zn2+ interaction of the zymogen. This is triggered by perturbation of the proMMP-2 molecule, but not by the reaction of the SH group of Cys73 with APMA. The 'activated' proMMP-2 (proMMP-2*) formed a new complex with TIMP-2 by binding to the N-terminal inhibitory domain of the inhibitor without processing the propeptide. Thus the APMA-treated proMMP-2*-TIMP-2 complex exhibited no gelatinolytic activity. In the presence of a small amount of free MMP-2, however, proMMP-2* in the complex was converted into the 65 kDa MMP-2 by proteolytic attack of MMP-2, but the complex did not exhibit gelatinolytic activity. The gelatinolytic activity detected after APMA treatment was solely derived from the activation of free proMMP-2. The removal of the propeptide of the proMMP-2* bound to TIMP-2 was also observed by MMP-3 (stromelysin 1), but not by MMP-1 (interstitial collagenase). MMP-3 cleaved the Asn80-Tyr81 bond of proMMP-2*. On the other hand, when MMP-3 was incubated with the proMMP-2-TIMP-2 complex, it bound to TIMP-2 and rendered proMMP-2 readily activatable by APMA. These results indicate that the blockage of TIMP-2 of the complex with an active MMP is essential for the activation of proMMP-2 when it is complexed with TIMP-2.
金属蛋白酶组织抑制剂（TIMP）-2通过每个分子的C末端结构域相互作用，与基质金属蛋白酶2（proMMP-2，前胶原酶A）的前体形成非共价复合物。我们从人子宫颈成纤维细胞培养基中分离出proMMP-2-TIMP-2复合物，并研究了4-氨基苯汞乙酸盐（APMA）对其激活过程的影响。用APMA处理该复合物会破坏酶原的Cys73-Zn2+相互作用，从而激活proMMP-2。这是由proMMP-2分子的扰动引发的，而不是Cys73的SH基团与APMA的反应。“激活的”proMMP-2（proMMP-2*）通过与抑制剂的N末端抑制结构域结合，在不处理前肽的情况下与TIMP-2形成新的复合物。因此，经APMA处理的proMMP-2*-TIMP-2复合物没有明胶溶解活性。然而，在存在少量游离MMP-2的情况下，复合物中的proMMP-2通过MMP-2的蛋白水解攻击转化为65 kDa的MMP-2，但该复合物没有明胶溶解活性。APMA处理后检测到的明胶溶解活性仅源于游离proMMP-2的激活。与TIMP-2结合的proMMP-2的前肽也可被MMP-3（基质溶解素1）去除，但不能被MMP-1（间质胶原酶）去除。MMP-3切割proMMP-2*的Asn80-Tyr81键。另一方面，当MMP-3与proMMP-2-TIMP-2复合物一起孵育时，它会与TIMP-2结合，并使proMMP-2易于被APMA激活。这些结果表明，当proMMP-2与TIMP-2复合时，复合物中TIMP-2的阻断对于proMMP-2的激活至关重要。
