Roswit W T, Halme J, Jeffrey J J
Arch Biochem Biophys. 1983 Aug;225(1):285-95. doi: 10.1016/0003-9861(83)90032-2.
A procollagenase from monolayer cultures of postpartum rat uterine cells has been purified. The crucial step in the purification is the binding of the procollagenase from crude, fetal bovine serum-containing culture medium to heparin-Sepharose, followed by elution with extremely low concentrations (5-10 nM) of dextran sulfate. Resultant eluates contain 8-10% procollagenase. Purification is completed by ion-exchange chromatography on DEAE-Sepharose, gel filtration on AcA-44, and chromatography on blue-Sepharose. Rat uterine procollagenase appears as a protein doublet of Mr approximately 58,000, as indicated by two polyacrylamide gel electrophoresis systems, by AcA-44 chromatography, and by equilibrium sedimentation ultracentrifugal analysis. The proenzyme forms are converted by trypsin to an active enzyme doublet of Mr approximately 48,000. Small amounts of active enzyme, which are often generated during the purification, are electrophoretically indistinguishable from trypsin-activated collagenase. Active collagenase can be separated from the zymogen forms by DEAE-Sepharose chromatography. The two forms of the proenzyme doublet can be partially separated by gel filtration on AcA-44 and preliminary analysis indicates each has equal collagenolytic activity. The amino acid analysis of rat uterine collagenase reveals it to be markedly different from two other vertebrate collagenases whose composition is known. The uterine proenzyme is unusually rich in glycine and in the hydroxy amino acids and is considerably more acidic than the human skin fibroblast collagenase, consistent with the different ion-exchange behavior of the two molecules. The specific activity of rat uterine collagenase at 37 degrees C is approximately 3000 micrograms collagen/min/mg, using native reconstituted guinea pig skin type I collagen fibrils as substrate. The enzyme cleaves denatured collagen, but fails to attack a variety of noncollagen proteins.
已从产后大鼠子宫细胞的单层培养物中纯化出一种前胶原酶。纯化过程中的关键步骤是将粗制的、含胎牛血清的培养基中的前胶原酶与肝素琼脂糖结合，然后用极低浓度（5 - 10 nM）的硫酸葡聚糖洗脱。所得洗脱液中含8 - 10%的前胶原酶。通过在DEAE - 琼脂糖上进行离子交换色谱、在AcA - 44上进行凝胶过滤以及在蓝色琼脂糖上进行色谱，完成纯化。如两种聚丙烯酰胺凝胶电泳系统、AcA - 44色谱以及平衡沉降超速离心分析所示，大鼠子宫前胶原酶表现为分子量约58,000的蛋白质双峰。前酶形式经胰蛋白酶转化为分子量约48,000的活性酶双峰。纯化过程中常产生的少量活性酶，在电泳上与胰蛋白酶激活的胶原酶无法区分。活性胶原酶可通过DEAE - 琼脂糖色谱与酶原形式分离。前酶双峰的两种形式可通过在AcA - 44上进行凝胶过滤部分分离，初步分析表明每种形式具有相同的胶原olytic活性。大鼠子宫胶原酶的氨基酸分析表明，它与另外两种已知组成的脊椎动物胶原酶明显不同。子宫前酶异常富含甘氨酸和羟基氨基酸，且比人皮肤成纤维细胞胶原酶酸性强得多，这与两种分子不同的离子交换行为一致。以天然重组的豚鼠皮肤I型胶原纤维为底物时，大鼠子宫胶原酶在37℃的比活性约为3000微克胶原/分钟/毫克。该酶能切割变性胶原，但不能作用于多种非胶原蛋白质。
