Mahaffey D, Yoo Y, Rechsteiner M
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
J Biol Chem. 1993 Oct 5;268(28):21205-11.
Xenopus egg extract is capable of supporting mitosis in vitro, which makes it ideal for biochemical analysis of the cell cycle. Since several studies have implicated the ubiquitin system in cell cycle progression, we have measured ubiquitin conjugation rates, proteolysis of ubiquitin-lysozyme conjugates, and rates of isopeptidase activity in cycling Xenopus egg extracts. Although ubiquitin conjugation in cytostatic factor arrested extract was half that in activated extract, there were no changes in rates of ubiquitin conjugation during the cell cycle. Ubiquitin conjugates are degraded by a 26 S ATP-stimulated protease. The ability of the 26 S protease to degrade ubiquitin-lysozyme conjugates and a fluorigenic peptide also remained constant across the cell cycle. In contrast to previously characterized systems, isopeptidase activity in Xenopus egg extract is energy-dependent. Glycerol gradient fractionation of Xenopus egg extract separated two ATP-dependent isopeptidases. On co-sedimented with the 26 S protease; the other sedimented slower and was not associated with any additional proteolytic activity. As found for rates of Ub conjugation and conjugate proteolysis, there was little or no variation in isopeptidase activity during the cell cycle.
非洲爪蟾卵提取物能够在体外支持有丝分裂，这使其成为细胞周期生化分析的理想选择。由于多项研究表明泛素系统参与细胞周期进程，我们测定了非洲爪蟾卵提取物在细胞周期中的泛素缀合率、泛素 - 溶菌酶缀合物的蛋白水解以及异肽酶活性。虽然在细胞静止因子阻滞提取物中的泛素缀合是活化提取物中的一半，但在细胞周期中泛素缀合率没有变化。泛素缀合物由一种26S ATP刺激的蛋白酶降解。26S蛋白酶降解泛素 - 溶菌酶缀合物和荧光肽的能力在整个细胞周期中也保持恒定。与之前表征的系统不同，非洲爪蟾卵提取物中的异肽酶活性是能量依赖的。非洲爪蟾卵提取物的甘油梯度分级分离出两种ATP依赖的异肽酶。一种与26S蛋白酶共沉降；另一种沉降较慢，且不与任何其他蛋白水解活性相关。正如泛素缀合率和缀合物蛋白水解率的情况一样，在细胞周期中异肽酶活性几乎没有变化。
