Evans R M, Ward D C, Fink L M
Proc Natl Acad Sci U S A. 1979 Dec;76(12):6235-9. doi: 10.1073/pnas.76.12.6235.
The distribution of plasma membrane-associated proteins was studied by using latex-filled phagolysosomes prepared from cultured mouse L-929 cells as a model of "inside-out" membrane. Proteins from 131I/lactoperoxidase-labeled phagolysosomes, phagolysosomes derived from 131I/lactoperoxidase-labeled cells, and phagolysosomes prepared from [35S]methionine metabolically labeled cells were analyzed by high-resolution two-dimensional gel electrophoresis. The gel patterns of iodinated proteins showed specific differences in the availability of membrane proteins to lactoperoxidase labeling between inside-out and right-side-out membranes. However, at least two prominent [35S]methionine-labeled proteins of approximately 60,000 and 100,000 daltons were available for iodination at both sides of the membrane. Partial proteolysis of the 100,000-dalton protein revealed that different peptides were iodinated when the iodination was performed on intact cells or on phagolysosomes, consisent with the idea that this protein spans the plasma membrane.
通过使用从培养的小鼠L-929细胞制备的充满乳胶的吞噬溶酶体作为“内外翻转”膜的模型，研究了质膜相关蛋白的分布。通过高分辨率二维凝胶电泳分析了来自131I/乳过氧化物酶标记的吞噬溶酶体、源自131I/乳过氧化物酶标记细胞的吞噬溶酶体以及由[35S]甲硫氨酸代谢标记细胞制备的吞噬溶酶体中的蛋白质。碘化蛋白质的凝胶图谱显示，内外翻转膜和正常方向膜之间，膜蛋白对乳过氧化物酶标记的可及性存在特定差异。然而，至少有两种约60,000和100,000道尔顿的突出的[35S]甲硫氨酸标记蛋白在膜的两侧均可被碘化。对100,000道尔顿蛋白的部分蛋白酶解显示，当在完整细胞或吞噬溶酶体上进行碘化时，不同的肽段会被碘化，这与该蛋白跨越质膜的观点一致。
