Bag J
Eur J Biochem. 1983 Sep 15;135(2):187-96. doi: 10.1111/j.1432-1033.1983.tb07636.x.
When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.
当鸡成肌细胞的原代培养物在高于其正常生长温度36 - 37摄氏度的温度下孵育时，蛋白质合成模式发生改变。这种热休克条件诱导大量蛋白质的强烈产生，这些蛋白质统称为“热休克蛋白”。热休克蛋白的合成在肌肉细胞产生的多种蛋白质产量没有显著下降的情况下实现。在单核分裂成肌细胞和终末分化的肌管中均观察到三种主要热休克多肽的合成，其分子量分别为81000、65000和25000。对成肌培养物合成的热诱导多肽进行二维电泳分离进一步证实，在成肌细胞和肌管中产生了相同的一组多肽，其分子量分别为65000（pI 6.0和5.5）、81000（pI 6.2）和25000（pI 5.6和5.3）。研究了蛋白质合成模式的变化对非多聚体细胞质mRNA - 蛋白质复合物（游离mRNP）的mRNA和蛋白质部分的影响。通过在蔗糖梯度中离心，从正常和热休克肌管培养物的核糖体后上清液中分离出沉降在20 - 35 S的游离mRNP复合物。从这些复合物中分离出的10 - 20S RNA组分在无细胞系统中刺激蛋白质合成。从热休克细胞获得的RNA组分似乎指导了所有三种主要热休克蛋白的合成。相反，当使用来自正常细胞游离mRNP复合物的RNA进行翻译时，未检测到这些多肽的合成。正常和热休克细胞的游离mRNP复合物在甲泛葡胺梯度中的浮力密度均为1.195 g/cm³。在从甲泛葡胺梯度中分离出的高度纯化的游离mRNP复合物中存在大量分子量为35000 - 105000的多肽。在来自正常和热休克肌管培养物的这些复合物中发现了相似的多肽组。然而,热休克细胞的游离mRNP复合物中分子量为65000的多肽的相对比例显著增加。二维凝胶电泳分析表明，该多肽与分子量为65000的热休克多肽表现出相似的电泳迁移特性。这些观察结果表明，对鸡肌管培养物进行热休克处理后，蛋白质合成模式的变化伴随着游离mRNP复合物的mRNA和蛋白质组成的改变。
