Baragi V M, Fliszar C J, Conroy M C, Ye Q Z, Shipley J M, Welgus H G
Department of Immunopathology, Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Ann Arbor, Michigan 48105.
J Biol Chem. 1994 Apr 29;269(17):12692-7.
In this study, we have used high resolution gel-filtration chromatography and measurements of Ki to compare the capacity of full-length native stromelysin, C-terminal truncated stromelysin (Phe100-Pro273), and matrilysin (the only metalloproteinase spontaneously lacking a C-terminal hemopexin-like domain) to bind to the tissue inhibitor of metalloproteinases (TIMP). While prostromelysin failed to bind TIMP, active stromelysin bound to the inhibitor avidly, exhibiting an affinity for TIMP (Ki = 8.3 x 10(-10) M) essentially identical to that of active interstitial collagenase as determined by competition experiments. C-terminal truncated stromelysin also formed a higher M(r) complex with TIMP which survived gel filtration. However, when truncated stromelysin was forced to compete with its full-length parent molecule for limiting amounts of TIMP, the full-length enzyme preferentially bound to the inhibitor. Indeed, binding studies indicated a Ki of 5.95 x 10(-9) M for the truncated variant's interaction with TIMP, only 14% as tight as that of full-length stromelysin. We also examined the interaction between TIMP and matrilysin, the only metalloproteinase which naturally lacks a C-terminal domain. Promatrilysin failed to bind the inhibitor. However, active matrilysin readily bound TIMP, forming a complex that resisted separation by gel filtration. When active matrilysin was forced to compete with truncated stromelysin for limiting amounts of TIMP, both enzymes appeared to complex the inhibitor with nearly equivalent efficacy. Indeed, active matrilysin exhibited a Ki for TIMP of 4.5 x 10(-9) M, essentially identical to that of truncated stromelysin. These data indicate that, as is true for collagenase, the C-terminal domain of stromelysin contributes significantly to its capacity to bind the physiologic inhibitor, TIMP. Furthermore, since stromelysin readily processes in vitro to a C-terminal truncated form, this enzyme species, as well as the full-length metalloproteinase matrilysin, may resist inhibition by TIMP in areas of active inflammation in vivo.
在本研究中，我们运用高分辨率凝胶过滤色谱法及 Ki 值测定，以比较全长天然基质溶解素、C 端截短的基质溶解素（Phe100 - Pro273）和基质溶素（唯一一种自然缺乏 C 端血红素结合蛋白样结构域的金属蛋白酶）与金属蛋白酶组织抑制剂（TIMP）结合的能力。原基质溶解素无法结合 TIMP，而活性基质溶解素能与该抑制剂紧密结合，通过竞争实验确定其对 TIMP 的亲和力（Ki = 8.3 x 10(-10) M）与活性间质胶原酶基本相同。C 端截短的基质溶解素也与 TIMP 形成了一种在凝胶过滤中稳定存在的更高相对分子质量复合物。然而，当截短的基质溶解素被迫与其全长亲本分子竞争有限量的 TIMP 时，全长酶优先与抑制剂结合。实际上，结合研究表明截短变体与 TIMP 相互作用的 Ki 值为 5.95 x 10(-9) M，仅为全长基质溶解素结合紧密程度的 14%。我们还研究了 TIMP 与基质溶素之间的相互作用，基质溶素是唯一一种天然缺乏 C 端结构域的金属蛋白酶。原基质溶素无法结合该抑制剂。然而，活性基质溶素能轻易结合 TIMP，形成一种在凝胶过滤中不易分离的复合物。当活性基质溶素被迫与截短的基质溶解素竞争有限量的 TIMP 时，两种酶似乎以几乎相同的效力与抑制剂形成复合物。实际上，活性基质溶素对 TIMP 的 Ki 值为 4.5 x 10(-9) M，与截短的基质溶解素基本相同。这些数据表明，与胶原酶情况相同，基质溶解素的 C 端结构域对其结合生理抑制剂 TIMP 的能力有显著贡献。此外，由于基质溶解素在体外容易加工成 C 端截短形式，这种酶以及全长金属蛋白酶基质溶素在体内活跃炎症区域可能会抵抗 TIMP 的抑制作用。
