Diettrich O, Gallert F, Hasilik A
Institut fur Physiologische Chemie der Philipps-Universitat, Marburg, Germany.
Eur J Cell Biol. 1996 Feb;69(2):99-106.
A major portion of the intracellular hydrolytic reactions are confined to lysosomes, whose membrane proteins take care of the proper lumenal milieu and the release of the degradation products. To obtain material for structural characterization of the transport proteins, we elaborated a procedure for isolation of lysosomal membranes and separation of their proteins in a two-dimensional electrophoresis. In a first step dense lysosomes were isolated from human placenta using a Percoll gradient. Subsequently, lysosomal membranes were purified by immunoadsorption. The procedure yielded mg-amounts of lysosomal membranes. Proteins associated with lysosomal membranes, acid beta-glucosidase, acetyl-coenzyme A:alpha-glucosaminide N-acetyltransferase, CD63/LIMP I, and h-lamp-2 were enriched approximately 300-fold as compared to the initial homogenate. Separation of the membrane proteins was achieved in a two-dimensional electrophoresis. The procedure is expected to yield material for structural studies on lysosomal membrane proteins with suspected defects in lysosomal storage diseases.
细胞内水解反应的主要部分局限于溶酶体，其膜蛋白负责维持合适的腔环境以及降解产物的释放。为了获得用于转运蛋白结构表征的材料，我们精心设计了一种从溶酶体膜中分离并通过二维电泳分离其蛋白质的方法。第一步，使用Percoll梯度从人胎盘中分离出致密的溶酶体。随后，通过免疫吸附纯化溶酶体膜。该方法产生了毫克量的溶酶体膜。与溶酶体膜相关的蛋白质、酸性β-葡萄糖苷酶、乙酰辅酶A：α-氨基葡萄糖苷N-乙酰转移酶、CD63/LIMP I和h-lamp-2与初始匀浆相比富集了约300倍。膜蛋白的分离通过二维电泳实现。该方法有望为研究溶酶体贮积症中疑似存在缺陷的溶酶体膜蛋白的结构提供材料。
