Plasma membrane-dependent activation of the 72-kDa type IV collagenase is prevented by complex formation with TIMP-2.
Plasma membrane-dependent activation of the 72-kDa type IV collagenase is prevented by complex formation with TIMP-2.
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发表时间：
1993-07
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通讯作者：
A. Strongin;B. Marmer;G. A. Grant;G. Goldberg
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作者：
A. Strongin;B. Marmer;G. A. Grant;G. Goldberg
人72-kDa IV型胶原酶（72 T4 Cl）作为酶原分泌，其可以通过与酶的羧基末端结构域的相互作用与金属蛋白酶TIMP-2的组织抑制剂形成特异性化学计量复合物。复合酶和游离酶都可以通过有机汞处理来激活。生理条件下72 T4 Cl活化的机制尚不清楚。在这里，我们描述了一个“质膜依赖性”激活的无标记的72 T4 Cl，并确定了第一个转换中间体作为一个64 kDa的物种从Asn 37-Leu肽键裂解的存在下，从12-O-十四烷酰佛波醇-13-乙酸诱导的HT 1080细胞的质膜。该反应对72 T4 Cl具有特异性，因为密切相关的酶原（92-kDa IV型胶原酶）在相同条件下对活化具有抗性。72T4Cl.TIMP-2复合物的形成在初始Asn 37-Leu切割水平抑制活化。TIMP-1的加入对该反应没有影响，但阻断Leu 38中间体向具有氨基末端Tyr 81的62-kDa活性酶的自催化转化。72 T4 Cl的膜依赖性活化在来自酶的羧基末端结构域的26-kDa肽的存在下被竞争性抑制。结果表明，酶的羧基末端结构域与膜相关组分的相互作用通过自蛋白水解机制引起酶活化的起始。
Human 72-kDa type IV collagenase (72T4Cl) is secreted as a proenzyme that can form a specific stoichiometric complex with the tissue inhibitor of metalloproteases TIMP-2 via interaction with the carboxyl-terminal domain of the enzyme. Both complexed and free enzymes can be activated by treatment with organomercurials. The mechanism of the 72T4Cl activation under physiological conditions is not known. Here we describe a "plasma membrane-dependent" activation of inhibitor-free 72T4Cl and identify the first conversion intermediate as a 64-kDa species resulting from cleavage of the Asn37-Leu peptide bond in the presence of plasma membranes from 12-O-tetradecanoylphorbol-13-acetate-induced HT1080 cells. This reaction is specific for 72T4Cl in that a closely related proenzyme (92-kDa type IV collagenase) is resistant to activation under the same conditions. Formation of the 72T4Cl.TIMP-2 complex inhibits activation at the level of the initial Asn37-Leu cleavage. Addition of TIMP-1 has no effect on this reaction, but blocks the autocatalytic conversion of the Leu38 intermediate into a 62-kDa active enzyme with an amino-terminal Tyr81. Membrane-dependent activation of 72T4Cl is competitively inhibited in the presence of a 26-kDa peptide derived from the carboxyl-terminal domain of the enzyme. The results suggest that interaction of the carboxyl-terminal domain of the enzyme with a membrane-associated component(s) causes initiation of enzyme activation through an autoproteolytic mechanism.