Membrane-bound ribosomes of myeloma cells. I. Preparation of free and membrane-bound ribosomal fractions. Assessment of the methods and properties of the ribosomes.
Membrane-bound ribosomes of myeloma cells. I. Preparation of free and membrane-bound ribosomal fractions. Assessment of the methods and properties of the ribosomes.
复制标题
骨髓瘤细胞的膜结合核糖体。 I.自由和膜结合的核糖体级分。评估核糖体的方法和特性。
DOI：
10.1083/jcb.67.1.1
复制
发表时间：
1975-10
影响因子：
7.8
通讯作者：
Vassalli, P
中科院分区：
文献类型：
--
作者：
Mechler, B;Vassalli, P
本文描述了一种细胞分离程序，该程序允许使用mopc21 （P3K）小鼠浆细胞瘤细胞培养，分离细胞质游离核糖体和膜结合核糖体，在没有相互交叉污染的部分中，并在多核糖体结构被完全保存。这是通过在不连续的蔗糖密度梯度上的沉淀实现的，其中两个核糖体种群向相反方向迁移。各种对照（电子显微镜、膜脂标记、分离部分的进一步再纯化）没有提供这些群体交叉污染的证据。然而，当分离前在细胞质萃取物中加入过量的用不同同位素标记的游离60S或40S亚基时，可以检测到膜组分捕获和/或吸附少量游离核糖体颗粒的可能性，特别是60S亚基；这可以通过使用含有0.15 M KC1的蔗糖梯度完全避免。EDTA处理后的膜组分几乎分离了所有的40S亚基，约70%的60S亚基。0.5 M KC1仅分离了10%的核糖体颗粒，这些颗粒由天然60S亚基和单核糖体组成，即结合颗粒在蛋白质合成中无活性。对游离核糖体和膜结合核糖体及其衍生的60S和40S核糖体亚基的CsC1浮力密度梯度分析表明，游离核糖体和膜结合核糖体颗粒具有相似的密度。
A cell fractionation procedure is described which allowed, by use of MOPC 21 (P3K) mouse plasmocytoma cells in culture, the separation of the cytoplasmic free and membrane-bound ribosomes in fractions devoid of mutual cross-contamination, and in which the polyribosomal structure was entirely preserved. This was achieved by sedimentation on a discontinuous sucrose density gradient in which the two ribosome populations migrate in opposite directions. A variety of controls (electron microscopy, labeling of membrane lipids, further repurification of the isolated fractions) provided no evidence of cross- contamination of these populations. However, when an excess of free 60S or 40S subunits, labeled with a different isotope, was added to the cytoplasmic extract before fractionation, the possibility of a small amount of trapping and/or adsorption of free ribosomal particles by the membrane fraction was detected, especially in the case of the 60S subunits; this could be entirely prevented by the use of sucrose gradients containing 0.15 M KC1. EDTA treatment of the membrane fraction detached almost all the 40S subunits, and about 70% of the 60S subunits. 0.5 M KC1 detached only 10% of the ribosomal particles, which consist of the native 60S subunits and the monoribosomes, i.e. the bound particles inactive in protein synthesis. Analysis in CsC1 buoyant density gradients of the free and membrane-bound polyribosomes and of their derived 60S and 40S ribosomal subunits showed that the free and membrane-bound ribosomal particles have similar densities.