Vincent A, Goldenberg S, Scherrer K
Eur J Biochem. 1981 Feb;114(2):179-93. doi: 10.1111/j.1432-1033.1981.tb05135.x.
EDTA dissociation of polyribosomes from duck erythroblasts allowed us to isolate the 15-S globin messenger ribonucleoproteins (mRNP) by sucrose gradient centrifugation or affinity chromatography on poly(U)-Sepharose or oligo(dT)-cellulose columns. Their protein composition was compared by one and two-dimensional electrophoresis in sodium dodecyl sulfate to the free 20-S mRNP containing the repressed fraction of globin mRNA [Vincent, A., Civelli, O., Maundrell, K., and Scherrer, K. (1980) Eur. J. Biochem. 112, 617--633]. The protein composition of the 15-S mRNP isolated by these methods in different ionic strength conditions, was characterized by a major 73 000-Mr polypeptide and seven minor polypeptides with Mr ranging from 45 000 to 68 000, all of which are slightly basic, and about five acidic ones in the 80 000--130 000-Mr range. All these are retained in the 15-S mRNP core particle isolated at 0.5 M KCl. At low ionic strength, in addition, a specific group of acidic polypeptides in the Mr range 35 000--105 000 was also found associated with globin mRNA. Oligo(dT)-cellulose chromatography of mRNP digested with ribonucleases A and T1 indicated that the 73 000-Mr major protein is bound to the poly(A) segment; some other proteins resolved as minor components interact with both the poly(A) and non-poly(A) regions of globin mRNA. Characterization of proteins interacting with the poly(A) segment of non-polyribosomal globin mRNA in 20-S free mRNP demonstrated the absence of the polyribosomal 73 000-Mr poly(A)-binding protein. Furthermore, it confirmed that the protein compositions of translatable polyribosomal and repressed free globin mRNP are very different. Indeed, the respective core (0.5 M KCl) particles contain only two possibly common polypeptides. The specificity of proteins associated with globin mRNA in two different functional states shown here supports the hypothesis of a role of mRNP proteins in translational control of mRNA.
用乙二胺四乙酸（EDTA）从鸭成红细胞中解离多核糖体，使我们能够通过蔗糖梯度离心或在聚（U）-琼脂糖或寡聚（dT）-纤维素柱上进行亲和层析来分离15-S珠蛋白信使核糖核蛋白（mRNP）。通过在十二烷基硫酸钠中进行一维和二维电泳，将其蛋白质组成与含有珠蛋白mRNA抑制部分的游离20-S mRNP进行比较[文森特，A.，奇维利，O.，蒙德雷尔，K.，和舍勒，K.（1980年）《欧洲生物化学杂志》112卷，617 - 633页]。在不同离子强度条件下通过这些方法分离的15-S mRNP的蛋白质组成，其特征是有一条主要的73000道尔顿的多肽和七条次要多肽，分子量在45000至68000之间，所有这些多肽都略带碱性，还有大约五条酸性多肽，分子量在80000至130000之间。所有这些都保留在在0.5M氯化钾中分离的15-S mRNP核心颗粒中。此外，在低离子强度下，还发现了一组分子量在35000至105000之间的特定酸性多肽与珠蛋白mRNA相关。用核糖核酸酶A和T1消化的mRNP进行寡聚（dT）-纤维素层析表明，73000道尔顿的主要蛋白质与聚（A）片段结合；一些作为次要成分分离的其他蛋白质与珠蛋白mRNA的聚（A）和非聚（A）区域都相互作用。对与20-S游离mRNP中无多核糖体的珠蛋白mRNA的聚（A）片段相互作用的蛋白质的表征表明，不存在多核糖体的73000道尔顿的聚（A）结合蛋白。此外，它证实了可翻译的多核糖体和受抑制的游离珠蛋白mRNP的蛋白质组成非常不同。实际上，各自的核心（0.5M氯化钾）颗粒仅包含两条可能共同的多肽。这里所示的与处于两种不同功能状态的珠蛋白mRNA相关的蛋白质的特异性支持了mRNP蛋白质在mRNA翻译控制中起作用的假说。
