Ubiquitin and ubiqutinated proteins in PC12 cells induced by NGF

观察神经生长因子(NGF)对PC12h细胞泛素免疫反应的影响。NGF处理的细胞胞核内泛素免疫反应增强。定量分析显示，NGF处理7d后，近20%的细胞有泛素免疫反应阳性核，此后其出现的频率增加。分别用放射免疫分析法(RIA)和酶联免疫吸附测定法(ELISA)测定细胞内游离泛素和多泛素链水平。对四种亚细胞组分进行了测量：胞核和细胞质的尿素和水溶性提取物。在NGF处理的细胞的水溶性胞浆提取物中观察到游离泛素的减少，但没有观察到同一组分中多泛素链的增加。在细胞核方面，经NGF处理后，水提物中多泛素链增加，游离泛素减少。经NGF处理后，尿素可溶的胞质提取物和核可溶尿素的胞质提取物中多泛素链的水平没有改变。这些结果表明，在NGF诱导PC12h细胞向神经元分化的过程中，核蛋白的多种泛素化水平增加。

Changes in ubiquitin-immunoreactivity after nerve growth factor (NGF) treatment were investigated in PC12h cells. Ubiquitin-immunoreactivity was increased in the nucleus of NGF-treated cells. The quantitiy analysis revealed that, after 7days of NGF treatment, almost 20% of cells had ubiquitin-immunoeractive nuclei and the frequency was increased thereafter. Levels of free ubiquitin and multi-ubiquitin chains were measured by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), respectively. Measurements were carried out for four subcellular fractions : urea-and water-soluble extracts of nuclei and cytoplasm. Decrease in free ubiquitin was observed in water-soluble cytoplasmic extracts of NGF-treated cells, though increase in multi-ubiquitin chains in the same fraction was not observed. As for nuclei, increase in multi-ubiquitin chains and concomitant decrease in free ubiquitin were found in the water-soluble extracts after NGF treatment. Lvels of multi-ubiquitin chains did not change in urea-soluble cytoplasmic extracts as well nuclear urea-soluble ones afer NGF treatment. These results indicated that multi-ubiquitination of nuclear proteins is increased during NGF-induced neuronal differentiation of PC12h cells.