Ubiquitin and ubiqutinated proteins in PC12 cells induced by NGF

研究了神经生长因子（NGF）作用后PC12h细胞泛素免疫反应性的变化。ngf处理的细胞核中泛素免疫反应性增强。定量分析显示，NGF处理7d后，近20%的细胞出现泛素免疫活性核，此后出现频率增加。用放射免疫法（RIA）和酶联免疫吸附法（ELISA）分别测定游离泛素和多泛素链的水平。测量进行了四个亚细胞部分：尿素和水溶性提取物的细胞核和细胞质。在ngf处理的细胞的水溶性细胞质提取物中，观察到游离泛素的减少，但在相同的部分中没有观察到多泛素链的增加。在细胞核方面，NGF处理后水溶性提取物中多泛素链增加，同时游离泛素减少。NGF处理后，脲溶性细胞质提取物和核脲溶性细胞质提取物中多泛素链的水平没有变化。这些结果表明，在ngf诱导的PC12h细胞神经元分化过程中，核蛋白的多泛素化增加。

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.