Roy L M, Swenson K I, Walker D H, Gabrielli B G, Li R S, Piwnica-Worms H, Maller J L
Howard Hughes Medical Institute, University of Colorado School of Medicine, Denver 80262.
J Cell Biol. 1991 May;113(3):507-14. doi: 10.1083/jcb.113.3.507.
Functional clam cyclin A and B proteins have been produced using a baculovirus expression system. Both cyclin A and B can induce meiosis I and meiosis II in Xenopus in the absence of protein synthesis. Half-maximal induction occurs at 50 nM for cyclin A and 250 nM for cyclin B. Addition of 25 nM cyclin A to activated Xenopus egg extracts arrested in the cell cycle by treatment with RNase or emetine activates cdc2 kinase to the normal metaphase level and stimulates one oscillatory cell cycle. High levels of cyclin A cause marked hyperactivation of cdc2 kinase and a stable arrest at the metaphase point in the cell cycle. Kinetic studies demonstrate the concentration of cyclin A added does not affect the 10 min lag period required for kinase activation or the timing of maximal activity, but does control the rate of deactivation of cdc2 kinase during exit from mitosis. In addition, exogenous clam cyclin A inhibits the degradation of both A- and B-type endogenous Xenopus cyclins. These results define a system for investigating the biochemistry and regulation of cdc2 kinase activation by cyclin A.
功能性蛤细胞周期蛋白A和B蛋白已通过杆状病毒表达系统产生。在没有蛋白质合成的情况下,细胞周期蛋白A和B都能诱导非洲爪蟾的减数分裂I和减数分裂II。细胞周期蛋白A的半最大诱导浓度为50 nM,细胞周期蛋白B为250 nM。向经核糖核酸酶或依米丁处理而停滞在细胞周期中的活化非洲爪蟾卵提取物中添加25 nM细胞周期蛋白A,可将cdc2激酶激活至正常中期水平,并刺激一个振荡细胞周期。高水平的细胞周期蛋白A会导致cdc2激酶显著过度激活,并在细胞周期的中期点稳定停滞。动力学研究表明,添加的细胞周期蛋白A浓度不影响激酶激活所需的10分钟延迟期或最大活性的时间,但确实控制了有丝分裂退出过程中cdc2激酶的失活速率。此外,外源蛤细胞周期蛋白A抑制非洲爪蟾内源性A类和B类细胞周期蛋白的降解。这些结果定义了一个用于研究细胞周期蛋白A对cdc2激酶激活的生物化学和调控的系统。