Stricklin G P, Bauer E A, Jeffrey J J, Eisen A Z
Biochemistry. 1977 Apr 19;16(8):1607-15. doi: 10.1021/bi00627a013.
Human skin procollagenase has been isolated, in pure form, from the medium of fibroblasts cultured in the presence or absence of added serum. Purification was achieved using a combination of cation-exchange (phosphocellulose or carboxymethylcellulose) and gel-filtration chromatography. Two forms (60 000 and 55 000 daltons) of the procollagenase were detected by electrophoresis in sodium dodecyl sulfatepolyacrylamide gels and could be separated by chromatography on Ultrogel AcA-44. Each form was converted to active enzyme by trypsin, producing species of 50 000 and 45 000 daltons, respectively. An autoactivation process also occurred, which yielded active enzyme without a detectable change in molecular weight. Procollagenase also was found in organ cultures of human skin but only when serum was added to the medium. This suggests that a serum-inhibitable proteolytic system is present in these cultures which, like trypsin, converts procollagenase to the active enzyme forms that can be isolated from serum-free organ culture medium. The collagenase species obtained from either fibroblast or organ culture medium were chromatographically and electrophoretically identical.
已从添加或未添加血清培养的成纤维细胞培养基中，以纯形式分离出人类皮肤原胶原酶。通过阳离子交换（磷酸纤维素或羧甲基纤维素）和凝胶过滤色谱法相结合实现纯化。在十二烷基硫酸钠-聚丙烯酰胺凝胶中进行电泳检测到两种形式（60000和55000道尔顿）的原胶原酶，并且可以通过在Ultrogel AcA - 44上进行色谱分离。每种形式都通过胰蛋白酶转化为活性酶，分别产生50000和45000道尔顿的产物。还发生了自激活过程，产生了活性酶，分子量没有可检测到的变化。在人类皮肤的器官培养物中也发现了原胶原酶，但仅当向培养基中添加血清时才会出现。这表明在这些培养物中存在一种血清抑制性蛋白水解系统，该系统与胰蛋白酶一样，将原胶原酶转化为可从无血清器官培养基中分离的活性酶形式。从成纤维细胞或器官培养基中获得的胶原酶种类在色谱和电泳上是相同的。
