Murphy G, Ward R, Hembry R M, Reynolds J J, Kühn K, Tryggvason K
Cell Physiology Department, Strangeways Research Laboratory, Cambridge, U.K.
Biochem J. 1989 Mar 1;258(2):463-72. doi: 10.1042/bj2580463.
The metalloproteinase 'gelatinase' stored in the granules of pig polymorphonuclear leucocytes has been purified in the latent form. The enzyme is secreted as an Mr 97,000 proenzyme that can be activated in the presence of 4-aminophenylmercuric acetate (APMA) by self-cleavage to generate lower-Mr species, of which an Mr 88,000 form was the most active. Trypsin-initiated activation generated different Mr gelatinases of much lower specific activity. Activation was slowed but not prevented by the presence of the tissue inhibitor of metalloproteinases, TIMP. The activated gelatinase formed a stable complex (Mr 144,000) with TIMP, in a Zn2+- and Ca2+-dependent manner, and complex formation was inhibited by the presence of the substrate gelatin. Similar to the human granulocyte gelatinase, the organomercurial-activated pig enzyme degraded gelatin and TCA and TCB fragments of type I collagen, as well as elastin and types IV and V collagen. The degradation of type IV collagen was shown, both by polyacrylamide-gel electrophoresis and by electron microscopic analysis, to generate 3/4 and 1/4 fragments as described for mouse tumour type IV collagenase. Furthermore, an antiserum raised to mouse type IV collagenase recognized the pig granulocyte gelatinase. An antiserum to the pig polymorphonuclear leucocyte gelatinase recognized other high-Mr gelatinases, including those from human granulocytes, pig monocytes and rabbit connective tissue cells, but not the Mr 72,000 enzyme from connective tissue cells. These data suggest that there are two distinct major forms of gelatinolytic activity that also cause specific cleavage of type IV collagen. These enzymes are associated with a wide variety of normal connective tissue and haemopoietic cells, as well as many tumour cells.
储存在猪多形核白细胞颗粒中的金属蛋白酶“明胶酶”已被纯化成潜伏形式。该酶以分子量为97,000的酶原形式分泌，在4-氨基苯基汞乙酸盐（APMA）存在下可通过自我切割被激活，产生分子量较低的形式，其中分子量为88,000的形式活性最高。胰蛋白酶引发的激活产生了比活性低得多的不同分子量的明胶酶。金属蛋白酶组织抑制剂TIMP的存在减缓了激活过程，但并未阻止激活。激活的明胶酶以锌离子和钙离子依赖的方式与TIMP形成稳定的复合物（分子量144,000），底物明胶的存在会抑制复合物的形成。与人类粒细胞明胶酶类似，有机汞激活的猪酶可降解明胶、I型胶原的三氯乙酸和三氯苯甲酸片段，以及弹性蛋白和IV型及V型胶原。聚丙烯酰胺凝胶电泳和电子显微镜分析均表明，IV型胶原的降解会产生如小鼠肿瘤IV型胶原酶所描述的3/4和1/4片段。此外，针对小鼠IV型胶原酶产生的抗血清可识别猪粒细胞明胶酶。针对猪多形核白细胞明胶酶产生的抗血清可识别其他高分子量的明胶酶，包括来自人类粒细胞、猪单核细胞和兔结缔组织细胞的明胶酶，但不能识别来自结缔组织细胞的分子量为72,000的酶。这些数据表明存在两种不同的主要明胶分解活性形式，它们也会导致IV型胶原的特异性切割。这些酶与多种正常结缔组织和造血细胞以及许多肿瘤细胞相关。
