REGULATION OF XENOPUS-LAEVIS DNA TOPOISOMERASE-I ACTIVITY BY PHOSPHORYLATION INVITRO
REGULATION OF XENOPUS-LAEVIS DNA TOPOISOMERASE-I ACTIVITY BY PHOSPHORYLATION INVITRO
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DOI：
10.1021/bi00409a014
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发表时间：
1988-05-03
期刊：
影响因子：
2.9
通讯作者：
BENBOW, RM
中科院分区：
文献类型：
--
作者：
KAISERMAN, HB;INGEBRITSEN, TS;BENBOW, RM
从非洲爪蟾卵巢中纯化出电泳纯的DNA拓扑异构酶I。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的最纯化的馏分揭示了一个单一的主要带在110 kDa和较少丰富的次要带集中在62 kDa。孵育的最纯化的馏分与固定化小牛肠碱性磷酸酶废除所有的DNA拓扑异构酶酶活性的时间依赖性反应。处理去磷酸化的X.带有X的左旋DNA拓扑异构酶I。Iaevis酪蛋白激酶II型活性和ATP使DNA拓扑异构酶活性恢复到高于在最纯化的级分中观察到的水平。使用最纯化的DNA拓扑异构酶I级分的体外标记实验，[γ- 32 P]ATP和酪蛋白激酶II型酶显示，110-和62-kDa条带均以大约摩尔比例磷酸化。磷酸化氨基酸分析表明，只有丝氨酸残基被磷酸化。磷酸化伴随着体外DNA拓扑异构酶活性的增加。DNA拓扑异构酶I的去磷酸化似乎阻断了初始酶/底物复合物的形成，这是基于在喜树碱存在下去磷酸化酶不能使DNA产生切口。我们的结论是X.左旋DNA拓扑异构酶I在分离时被部分磷酸化，并且这种磷酸化对于体外酶活性的表达是必需的。酪蛋白激酶II型活性的基础上，重新激活去磷酸化的DNA拓扑异构酶I的能力，我们推测，这种激酶可能有助于DNA拓扑异构酶I活性的生理调节。
DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, [.gamma.-32P]ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme.sbd.substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. We conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, we speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.