A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro.
A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro.
复制标题
原位和体外磷酸化兔网织红细胞核糖体蛋白的比较。
DOI：
10.1021/bi00648a025
复制
发表时间：
1976
期刊：
影响因子：
2.9
通讯作者：
G. G. Porter
中科院分区：
文献类型：
--
作者：
J. Traugh;G. G. Porter
对完整细胞中磷酸化的核糖体蛋白与经纯化蛋白激酶和 [gamma-32P]ATP 体外修饰后从核糖体亚基中分离的蛋白质进行了比较。当完整的网织红细胞在含有放射性无机磷酸盐的营养培养基中孵育 2 小时时，使用二维聚丙烯酰胺凝胶电泳，然后在含有十二烷基硫酸钠的第三步中进行电泳，将一种磷酸化蛋白质鉴定为 40S 核糖体成分。通过二维电泳观察到，这种蛋白质含有与核糖体蛋白质相关的总放射性的 99%，除了几种磷酸化状态外，还发现其处于非磷酸化形式。这些状态因蛋白质上附着的磷酰基的数量而异。相同的 40S 蛋白在体外被兔网织红细胞的三种 cAMP 调节蛋白激酶修饰。与 40S 亚基相关的另外两种蛋白质被原位磷酸化。这些蛋白质作为对称双联体迁移，并且 40S 亚基中含有不到 1% 的放射性磷酸盐。将全细胞与标记的磷酸盐一起孵育后，通过圆盘凝胶电泳观察到许多与 60S 亚基相关的磷酸化蛋白质。这些蛋白质不与先前鉴定的核糖体蛋白质一起迁移，并且存在的量不足以被鉴定为核糖体结构蛋白质。大亚基中的蛋白质在体外被 cAMP 调节的蛋白激酶和 ATP 修饰，并且这些修饰的蛋白质与已知的核糖体蛋白质一起迁移。然而，这种磷酸化尚未显示在完整细胞中发生。
A comparison has been made between the ribosomal proteins phosphorylated in intact cells and proteins isolated from ribosomal subunits after modification in vitro by purified protein kinases and [gamma-32P]ATP. When intact reticulocytes were incubated for 2 h in a nutritional medium containing radioactive inorganic phosphate, one phosphorylated protein was identified as a 40S ribosomal component using two-dimensional polyacrylamide gel electrophoresis followed by electrophoresis in a third step containing sodium dodecyl sulfate. This protein, containing 99% of the total radioactivity associated with ribosomal proteins as observed by two-dimensional electrophoresis, is found in a nonphosphorylated form in addition to several phosphorylated states. These states differ by the number of phosphoryl group attached to the protein. The same 40S protein is modified in vitro by the three cAMP-regulated protein kinases from rabbit reticulocytes. Two additional proteins associated with the 40S subunit are phosphorylated in situ. These proteins migrate as a symmetrical doublet, and contain less than 1% of the radioactive phosphate in the 40S subunit. A number of phosphorylated proteins associated with 60S subunits are observed by disc gel electrophoresis after incubation of whole cells with labeled phosphate. These proteins do not migrate with previously identified ribosomal proteins and are not present in sufficient amounts to be identified as ribosomal structural proteins. Proteins in the large subunit are modified in vitro by cAMP-regulated protein kinases and ATP, and these modified proteins migrate with known ribosomal proteins. However, this phosphorylation has not been shown to occur in intact cells.