Characterization of proteins in detergent-resistant membrane complexes from Madin-Darby canine kidney epithelial cells
Characterization of proteins in detergent-resistant membrane complexes from Madin-Darby canine kidney epithelial cells
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DOI：
10.1021/bi00049a031
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发表时间：
1995-12-12
期刊：
影响因子：
2.9
通讯作者：
Brown, DA
中科院分区：
文献类型：
--
作者：
Melkonian, KA;Chu, T;Brown, DA
我们之前分离出了耐去污剂膜复合物 (DRM)，在冰上用 Triton X-100 提取 Madin-Darby 犬肾细胞后，该膜复合物未溶解。这些复合物富含鞘糖脂、胆固醇和糖基磷脂酰肌醇 (GPI) 锚定蛋白。在这项研究中，我们检查了 DRM 的蛋白质组成，并进一步表征了这些结构的洗涤剂溶解度。在 DRM 中回收了 8 到 10 种细胞表面蛋白，包括来自顶膜和基底外侧膜的蛋白。然而，大多数 DRM 蛋白并未暴露于整个细胞的表面，我们也没有检测到 Sargiacomo 等人描述的细胞表面蛋白复合物。在一项类似的研究中 [Sargiacomo, M., et al. (1993) J.细胞生物学。 122、789-807]。 DRM 中的几乎所有蛋白质均在 30 摄氏度以上的温度下被 Triton X-100 或冰上的辛基葡萄糖苷溶解。相比之下，GPI 锚定蛋白（胎盘碱性磷酸酶）在 10 摄氏度下提取后大部分被 Triton X-100 溶解。该蛋白在首次递送到质膜时不溶于冰冷的 Triton X-100，并且在合成后至少保持 6 小时。在 37 摄氏度下用 Triton X-100 提取后，DRM 中的一部分脂质仍然不溶。DRM 脂质不被辛基葡萄糖苷溶解，表明这种洗涤剂选择性从 DRM 中提取蛋白质。
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 oxtyl glucoside, suggesting that this detergent selectivity extracts proteins from DRMs.