Burkhardt J, Huber L A, Dieplinger H, Blocker A, Griffiths G, Desjardins M
European Molecular Biology Laboratory, Heidelberg.
Electrophoresis. 1995 Dec;16(12):2249-57. doi: 10.1002/elps.11501601357.
Phagosomes are the organelles formed de novo in a variety of cells by the internalization of large particulate materials, including a wide range of pathogenic microorganisms. We present here a systematic approach that can be used to study the polypeptide composition of phagosomes/phagolysosomes and to yield analytical information on the characteristics of their proteins. A density shift approach was used to isolate pure preparations of phagosomes filled with low density latex beads from mouse J774 and human U937 macrophages. High resolution two-dimensional (2-D) gel electrophoresis was performed to generate a map of the overall [35S]methionine-labeled protein profile of the isolated phagosomes. The resulting map showed the minimal presence of over 200 polypeptides, indicating the complexity of this organelle. Comigration experiments showed that several phagosome polypeptides, among them several known proteins, are shared by the two species. Extraction with Triton X-114 and sodium carbonate was performed to distinguish between membrane and soluble proteins, and sensitivity to a panel of proteases was measured to identify proteins exposed on the cytoplasmic face of the phagosome membrane. The general value of the 2-D gel approach in the mapping of organelle proteins is discussed.
吞噬体是各种细胞通过内化大颗粒物质（包括多种致病微生物）而重新形成的细胞器。我们在此介绍一种系统方法，可用于研究吞噬体/吞噬溶酶体的多肽组成，并获取有关其蛋白质特征的分析信息。采用密度转移方法从小鼠J774和人U937巨噬细胞中分离出充满低密度乳胶珠的吞噬体纯制剂。进行高分辨率二维（2-D）凝胶电泳，以生成分离出的吞噬体的整体[35S]甲硫氨酸标记蛋白质谱图。所得图谱显示存在至少200种多肽，表明该细胞器的复杂性。共迁移实验表明，两种细胞共有几种吞噬体多肽，其中包括几种已知蛋白质。用Triton X-114和碳酸钠进行提取以区分膜蛋白和可溶性蛋白，并测定对一组蛋白酶的敏感性以鉴定暴露于吞噬体膜细胞质面的蛋白质。讨论了二维凝胶方法在细胞器蛋白质图谱绘制中的一般价值。
