Amar-Costesec A, Hortsch M, Turu C
International Institute of Cellular and Molecular Pathology, Université Catholique de Louvain, Brussels, Belgium.
Biol Cell. 1988;62(3):281-8.
We describe a procedure for disassembling rat liver rough microsomes, which allows the purification of the rough endoplasmic reticulum (ER) membrane. Membrane-bound ribosomes and adsorbed proteins are first detached by washing rough microsomes with 5 mM Na-pyrophosphate. In a second step, the vesicle membrane is opened by digitonin, with concomitant release of the luminal content. The purification is monitored at each step by electron microscopy, and by assaying chemical constituents (protein, phospholipid, RNA) and marker enzymes for the main subcellular organelles. The final membrane preparation is representative of the ER, since it contains 24.1% of the liver glucose 6-phosphatase with a relative specific activity of 14.2. Contaminants represent less than 5% of its protein content. SDS-polyacrylamide gel electrophoresis, followed by immunoblot analysis, reveals that the ribophorins I and II, two established markers of the rough (d) domain are still present in the final membrane preparation. It also contains the docking protein (or signal recognition particle receptor) and protein disulfide isomerase, and has conserved the functional capacity to remove co- and post-translationally the signal peptide of pre-secretory proteins. The membrane preparation is suitable for studies on the polypeptide composition of the d domain.
我们描述了一种拆解大鼠肝脏粗面微粒体的方法，该方法可用于纯化粗面内质网（ER）膜。首先用5 mM焦磷酸钠洗涤粗面微粒体，使膜结合核糖体和吸附蛋白脱离。第二步，用洋地黄皂苷打开囊泡膜，同时释放腔内成分。在每一步通过电子显微镜以及分析主要亚细胞器的化学成分（蛋白质、磷脂、RNA）和标记酶来监测纯化过程。最终的膜制剂代表内质网，因为它含有肝脏葡萄糖6-磷酸酶的24.1%，相对比活性为14.2。污染物占其蛋白质含量的不到5%。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，随后进行免疫印迹分析，结果显示粗面（d）结构域的两个既定标记物核糖体结合糖蛋白I和II仍存在于最终的膜制剂中。它还含有对接蛋白（或信号识别颗粒受体）和蛋白质二硫键异构酶，并且保留了在翻译前和翻译后去除分泌前蛋白信号肽的功能能力。该膜制剂适用于研究d结构域的多肽组成。
