Fioretti W C, Davis D F, Ledford B E
Biochim Biophys Acta. 1979 Aug 29;564(1):79-89. doi: 10.1016/0005-2787(79)90190-4.
The analysis of translational efficiencies of specific mRNAs requires a determination of the polyribosome size. The appropriate value to use in such calculations is the number-average size. A method is described for accurately measuring the number-average size of total and of specific protein synthesizing polyribosomes using isokinetic sucrose density gradients and 125I-labeled antibodies. By this method, we demonstrated that albumin synthesizing polyribosomes from a serum albumin secreting mouse hepatoma cell line exist over a broad range from trimers to 20-mers (mean 6-10). The specificity of antibody interaction with polyribosomes was demonstrated using cells not synthesizing mouse serum albumin, and by demonstrating that 125I-anti ovalbumin does not bind to mouse hepatoma polyribosomes. Treatment of the mouse hepatoma cells with 1 MUM cycloheximide shifted practically all of the monomers into polyribosomes resulting in an increase in the number-average size of the albumin synthesizing polyribosomes. Cycloheximide treatment, however, did not eliminate the size heterogeneity in the albumin synthesizing polyribosomes.
对特定mRNA翻译效率的分析需要测定多核糖体的大小。此类计算中适用的合适值是数均大小。本文描述了一种使用等速蔗糖密度梯度和125I标记抗体精确测量总多核糖体和特定蛋白质合成多核糖体数均大小的方法。通过该方法，我们证明了来自分泌血清白蛋白的小鼠肝癌细胞系的白蛋白合成多核糖体存在于从三聚体到二十聚体的广泛范围内（平均6 - 10）。使用不合成小鼠血清白蛋白的细胞，并通过证明125I - 抗卵清蛋白不与小鼠肝癌多核糖体结合，证实了抗体与多核糖体相互作用的特异性。用1 μM环己酰亚胺处理小鼠肝癌细胞实际上使所有单体都进入了多核糖体，导致白蛋白合成多核糖体的数均大小增加。然而，环己酰亚胺处理并未消除白蛋白合成多核糖体中的大小异质性。
