Phillips W F, McConkey E H
J Biol Chem. 1976 May 25;251(10):2876-81.
Total protein was released from isolated HeLa cell nucleoli by guanidine hydrochloride, purified by cesium chloride density gradient centrifugation, and analyzed by two-dimensional polyacrylamide gel electrophoresis. Conditions of electrophoresis restricted attention to proteins that are positively charged at pH 8.6. Most of the major nucleolar protein spots co-electrophoresed with ribosomal proteins; the majority of ribosomal proteins from both the large and small ribosomal subunits were represented. Several proteins found in association with polysomes but not on ribosomal subunits and several proteins unique to the nucleolus were also identified in these nucleolar protein patterns. In order to determine whether the ribosomal proteins found in the nucleolus represented sizable pools of ribosomal proteins, or merely ribosomal proteins contained in the preribosomal particles, [35S]methionine-labeled nucleoli were mixed with [3H]methionine-labeled polysomes. From analysis of isotopic ratios in individual protein spots it was possible to determine the stoidchiometry of individual ribosomal proteins in the nucleolus relative to their complement on cytoplasmic ribosomes. All but a few proteins exhibited relative nucleolar stoichiometry values of approximately one, indicating that there are not significant pools of most ribosomal proteins in isolated nucleoli.
通过盐酸胍从分离的HeLa细胞核仁中释放总蛋白，经氯化铯密度梯度离心纯化，并用二维聚丙烯酰胺凝胶电泳进行分析。电泳条件将研究重点限制在pH 8.6时带正电荷的蛋白质上。大多数主要核仁蛋白斑点与核糖体蛋白共电泳；大小核糖体亚基的大多数核糖体蛋白都有呈现。在这些核仁蛋白图谱中还鉴定出了几种与多核糖体相关但不在核糖体亚基上的蛋白质以及几种核仁特有的蛋白质。为了确定在核仁中发现的核糖体蛋白是代表相当数量的核糖体蛋白库，还是仅仅是前核糖体颗粒中所含的核糖体蛋白，将[35S]甲硫氨酸标记的核仁与[3H]甲硫氨酸标记的多核糖体混合。通过分析单个蛋白斑点中的同位素比率，可以确定核仁中单个核糖体蛋白相对于其在细胞质核糖体上的互补物的化学计量。除少数几种蛋白质外，所有蛋白质的相对核仁化学计量值都约为1，这表明在分离的核仁中大多数核糖体蛋白不存在显著的蛋白库。
