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首页> 美国卫生研究院文献>The Journal of Biophysical and Biochemical Cytology >Membrane-bound ribosomes of myeloma cells. I. Preparation of free and membrane-bound ribosomal fractions. Assessment of the methods and properties of the ribosomes
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Membrane-bound ribosomes of myeloma cells. I. Preparation of free and membrane-bound ribosomal fractions. Assessment of the methods and properties of the ribosomes

机译：骨髓瘤细胞的膜结合核糖体。 I.制备游离的和膜结合的核糖体级分。核糖体的方法和性质的评估

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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.
机译：描述了一种细胞分离程序，该程序允许使用培养中的MOPC 21（P3K）小鼠浆细胞瘤细胞，将细胞质游离和膜结合核糖体分离成没有相互交叉污染的组分，并且其中的多核糖体结构为完全保留。这是通过在不连续的蔗糖密度梯度上进行沉降来实现的，其中两个核糖体群体沿相反的方向迁移。各种对照（电子显微镜检查，膜脂质标记，分离部分的进一步纯化）都没有提供这些种群交叉污染的证据。但是，在分级分离之前，如果将过量的60S或40S亚基标记有不同的同位素的游离亚基添加到细胞质提取物中，则会检测到膜部分会少量捕获和/或吸附游离核糖体颗粒，尤其是在60S亚单位的情况下；这可以通过使用含有0.15 M KC1的蔗糖梯度完全防止。膜部分的EDTA处理分离了几乎所有40S亚基，以及约70％的60S亚基。 0.5 M KC1仅分离了10％的核糖体颗粒，该颗粒由天然60S亚基和单核糖体组成，即在蛋白质合成中没有活性的结合颗粒。游离和膜结合的核糖体及其衍生的60S和40S核糖体亚基的CsC1浮力密度梯度分析表明，游离和膜结合的核糖体颗粒具有相似的密度。

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