Takiguchi M, Mori M, Tatibana M
J Biochem. 1985 May;97(5):1447-59. doi: 10.1093/oxfordjournals.jbchem.a135199.
A procedure is described for the preparation of free and membrane-bound polysomes from rat liver. The procedure involves: differential centrifugation of liver homogenate to separate free and membrane-bound polysomes; treatment of the membrane-bound polysome fraction with a detergent to release bound polysomes from membranes; and magnesium precipitation of both classes of polysomes. Free and bound polysomes prepared in this manner were essentially undegraded and highly active in cell-free protein synthesis. The recovery of polysomes was nearly quantitative and the distribution between the free and membrane-bound state was 41 and 59%, respectively. Polypeptides synthesized in vitro by the free and membrane-bound polysomes were quite different. The majority (81-84%) of mRNA activities of two secretory proteins (albumin and transferrin) were recovered in the membrane-bound polysomes, whereas the majority (81-85%) of mRNA activities of two cytosolic [aldolase B, EC 4.1.2.13, and argininosuccinate synthetase, EC 6.3.4.5], one mitochondrial [ornithine carbamoyltransferase, EC 2.1.3.3] and one peroxisomal [catalase, EC 1.11.1.6] proteins were recovered in the free polysomes. A polysome class synthesizing ornithine carbamoyltransferase was purified 42-fold from the free polysomes by immunoprecipitation. The procedure is rapid (4-5 h) and reproducible, and provides a nearly quantitative means of separating the two classes of polysomes.
本文描述了一种从大鼠肝脏制备游离和膜结合多核糖体的方法。该方法包括：对肝脏匀浆进行差速离心以分离游离和膜结合多核糖体；用去污剂处理膜结合多核糖体部分以从膜上释放结合的多核糖体；以及对两类多核糖体进行镁沉淀。以这种方式制备的游离和结合多核糖体基本上未降解，并且在无细胞蛋白质合成中具有高活性。多核糖体的回收率几乎是定量的，游离态和膜结合态之间的分布分别为41%和59%。由游离和膜结合多核糖体体外合成的多肽有很大不同。两种分泌蛋白（白蛋白和转铁蛋白）的大部分mRNA活性（81 - 84%）存在于膜结合多核糖体中，而两种胞质蛋白[醛缩酶B，EC 4.1.2.13，和精氨琥珀酸合成酶，EC 6.3.4.5]、一种线粒体蛋白[鸟氨酸氨甲酰转移酶，EC 2.1.3.3]和一种过氧化物酶体蛋白[过氧化氢酶，EC 1.11.1.6]的大部分mRNA活性（81 - 85%）存在于游离多核糖体中。通过免疫沉淀从游离多核糖体中纯化出合成鸟氨酸氨甲酰转移酶的一类多核糖体，纯化倍数为42倍。该方法快速（4 - 5小时）且可重复，提供了一种几乎定量分离两类多核糖体的方法。
