Melkonian K A, Chu T, Tortorella L B, Brown D A
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794-5215, USA.
Biochemistry. 1995 Dec 12;34(49):16161-70. doi: 10.1021/bi00049a031.
We previously isolated detergent-resistant membrane complexes (DRMs) that were not solubilized after extraction of Madin-Darby canine kidney cells with Triton X-100 on ice. The complexes were rich in glycosphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins. In this study, we examined the protein composition of DRMs and further characterized the detergent solubility of these structures. Eight to ten cell-surface proteins, including proteins from both apical and basolateral membranes, were recovered in DRMs. Most DRM proteins, however, were not exposed to the surface of whole cells, and we did not detect the complex of cell-surface proteins described by Sargiacomo et al. in a similar study [Sargiacomo, M., et al. (1993) J. Cell Biol. 122, 789-807]. Almost all proteins in DRMs were solubilized by Triton X-100 at temperatures above 30 degrees C or by octyl glucoside on ice. In contrast, a GPI-anchored protein, placental alkaline phosphatase, was mostly solubilized by Triton X-100 after extraction at 10 degrees C. This protein was insoluble in ice-cold Triton X-100 when first delivered to the plasma membrane and remained so for at least 6 h after synthesis. A fraction of the lipids in DRMs remained insoluble after extraction with Triton X-100 at 37 degrees C. DRM lipids were not solubilized by octyl glucoside, suggesting that this detergent selectively extracts proteins from DRMs.
我们之前分离出了耐去污剂膜复合物（DRM），在用冰上的Triton X-100提取麦迪逊-达比犬肾细胞后，这些复合物未被溶解。这些复合物富含糖鞘脂、胆固醇和糖基磷脂酰肌醇（GPI）锚定蛋白。在本研究中，我们检测了DRM的蛋白质组成，并进一步表征了这些结构的去污剂溶解性。在DRM中回收了8至10种细胞表面蛋白，包括来自顶端和基底外侧膜的蛋白。然而，大多数DRM蛋白并未暴露于完整细胞的表面，并且我们未检测到Sargiacomo等人在一项类似研究中描述的细胞表面蛋白复合物[Sargiacomo, M., et al. (l993) J. Cell Biol. 122, 789 - 807]。几乎所有DRM中的蛋白在30摄氏度以上的温度下可被Triton X-100溶解，或者在冰上可被辛基葡糖苷溶解。相比之下，一种GPI锚定蛋白，胎盘碱性磷酸酶，在10摄氏度提取后大多可被Triton X-100溶解。当该蛋白首次转运至质膜时，它在冰冷的Triton X-100中不溶解，并且在合成后至少6小时内一直保持这种状态。在37摄氏度用Triton X-100提取后，DRM中的一部分脂质仍不溶解。DRM脂质不能被辛基葡糖苷溶解，这表明这种去污剂选择性地从DRM中提取蛋白质。
