Fujimoto N, Ward R V, Shinya T, Iwata K, Yamashita K, Hayakawa T
Biotechnology Section, Research Laboratories I, Fuji Chemical Industries Ltd., Toyama, Japan.
Biochem J. 1996 Feb 1;313 ( Pt 3)(Pt 3):827-33. doi: 10.1042/bj3130827.
By immunoreactivity analysis using monoclonal antibodies, we showed that the C-terminal domain [R415-631; R is residue] of progelatinase A [pro-matrix metalloproteinase-2 (proMMP-2); EC 3.4.24.24] affected the immunoreactivity of a one-step sandwich enzyme immunoassay (sandwich EIA) for tissue inhibitor of metalloproteinases-2 (TIMP-2) in exactly the same way as does proMMP-2 [Fujimoto, Zhang, Iwata, Shinya, Okada and Hayakawa (1993) Clin. Chim. Acta 220, 31-45], confirming that the C-terminal domain ("tail" portion of TIMP-2 participates in the binding with the C-terminal domain of proMMP-2. We also demonstrated that not only the C-terminal domain but also the N-terminal domain (R1-417) of proMMP-2 bound to TIMP-2 in a 1:1 molar ratio. The binding of each individual domain to TIMP-2, however, was weak enough that either domain could be fully replaced by proMMP-2 through the same binding sites as does proMMP-2, and also that the high-order structure of proMMP-2 allows a more stable binding to TIMP-2. We further confirmed that TIMP-2 complexed with the N-terminal domain of pro-MMP-2 had fully inhibitory activity against the collagenolytic activity of MMP-1. We also demonstrated that either the interstitial collagenase-TIMP-2 complex or the gelatinase B(MMP-9)-TIMP-2 complex was able to form a ternary complex with proMMP-2 in a 1:1 molar ratio, clearly indicating that there are two distinct binding sites, one specific for proMMP-2 complex, but the binding seemed to be less stable than the binding with TIMP-2 alone. Even in the presence of a 10-fold molar excess of the N-terminal domain, ternary complex formation was not observed between the N-terminal domain and the MMP-9--TIMP-2 complex. These clear differences might be ascribed to some significant conformational change(s) evoked in the TIMP-2 molecule, or hindrance of a part of the N-terminal domain binding site of TIMP-2 by complex formation with MMP-9.
通过使用单克隆抗体进行免疫反应性分析，我们发现前明胶酶A [前基质金属蛋白酶-2（proMMP-2）；EC 3.4.24.24]的C末端结构域[R415 - 631；R为残基]对金属蛋白酶组织抑制剂-2（TIMP-2）的一步夹心酶免疫测定（夹心EIA）的免疫反应性的影响与proMMP-2完全相同[藤本、张、岩田、 Shinya、冈田和早川（1993年）《临床化学学报》220，31 - 45]，证实C末端结构域（TIMP-2的“尾部”部分）参与与proMMP-2的C末端结构域的结合。我们还证明，不仅proMMP-2的C末端结构域，而且其N末端结构域（R1 - 417）也以1:1的摩尔比与TIMP-2结合。然而，每个单独结构域与TIMP-2的结合都足够弱，以至于任何一个结构域都可以被proMMP-2通过与proMMP-2相同的结合位点完全取代，并且proMMP-2的高级结构允许与TIMP-2形成更稳定的结合结构。我们进一步证实，与pro-MMP-2的N末端结构域复合的TIMP-2对MMP-1的胶原分解活性具有完全抑制活性。我们还证明，间质胶原酶-TIMP-2复合物或明胶酶B（MMP-9）-TIMP-2复合物都能够以1:1的摩尔比与proMMP-2形成三元复合物，清楚地表明存在两个不同的结合位点，一个对proMMP-2复合物具有特异性，但这种结合似乎比仅与TIMP-2的结合更不稳定。即使存在10倍摩尔过量的N末端结构域，在N末端结构域与MMP-9 - TIMP-2复合物之间也未观察到三元复合物的形成。这些明显的差异可能归因于TIMP-2分子中引起的一些显著构象变化，或者由于与MMP-9形成复合物而对TIMP-2的N末端结构域结合位点的一部分产生的阻碍。
