Stenn K S, Link R, Moellmann G, Madri J, Kuklinska E
Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Invest Dermatol. 1989 Aug;93(2):287-90. doi: 10.1111/1523-1747.ep12277593.
Dispase, a neutral protease isolated from culture filtrates of Bacillus polymyxa, has proven to be a rapid, effective, but gentle agent for separating intact epidermis from the dermis and intact epithelial sheets in culture from the substratum. In both cases it effects separation by cleaving the basement membrane zone region while preserving the viability of the epithelial cells. Because it is not known what or where in the basement membrane zone Dispase cleaves, we set up studies to define its substrate specificity. Using purified basement membrane components and sodium dodecyl sulfate-polyacrylamide gel electrophoresis we show that Dispase cleaves fibronectin and type IV collagen, but not laminin, type V collagen, serum albumin, or transferrin. The action of Dispase on collagen appears to be selective for type IV collagen in that several stable degradation products are formed, whereas the enzyme degrades type I collagen only minimally. In newborn human skin, as seen by electron microscopy, Dispase removes the lamina densa, rich in type IV collagen, but preserves the anchoring fibrils (structures known to contain type VII collagen) and the epidermal cells. Because its action is so selective, it suggests that Dispase can serve as a powerful tool for dissecting epithelial-mesenchymal interactions.
分散酶是一种从多粘芽孢杆菌培养滤液中分离出来的中性蛋白酶，已被证明是一种快速、有效且温和的试剂，可用于从真皮中分离完整的表皮，以及从培养物的基质中分离完整的上皮细胞片层。在这两种情况下，它通过切割基底膜区来实现分离，同时保持上皮细胞的活力。由于尚不清楚分散酶在基底膜区切割的是什么物质以及切割位置，我们开展了研究以确定其底物特异性。利用纯化的基底膜成分和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳，我们发现分散酶可切割纤连蛋白和IV型胶原，但不切割层粘连蛋白、V型胶原、血清白蛋白或转铁蛋白。分散酶对胶原的作用似乎对IV型胶原具有选择性，因为会形成几种稳定的降解产物，而该酶对I型胶原的降解作用极小。在新生儿人类皮肤中，通过电子显微镜观察发现，分散酶去除了富含IV型胶原的致密板层，但保留了锚定原纤维（已知含有VII型胶原的结构）和表皮细胞。由于其作用具有高度选择性，这表明分散酶可作为剖析上皮 - 间充质相互作用的有力工具。
