Bosser R, Roig J, Itare E, Bachs O
Department of Cell Biology, Faculty of Medicine, University of Barcelona, Spain.
Biochem Biophys Res Commun. 1994 Jul 29;202(2):984-91. doi: 10.1006/bbrc.1994.2026.
Casein kinase 2 was released from rat liver cells nuclei by digestion with DNase I plus RNase A. This treatment also released three major substrates of 50, 40-42, and 37 kDa. Casein kinase 2 and substrates were also extracted by DNase or RNase separately. However, in DNase extracts only the 37 kDa protein was phosphorylated by casein kinase 2, whereas in RNase extracts all three substrates were phosphorylated. When the DNase extracts were subsequently treated with RNase the 40-42 substrates were then phosphorylated, indicating that their interaction with RNA prevents their phosphorylation by casein kinase 2. The ratio of B: alpha subunits of casein kinase 2 present in the nuclease extracts was higher than that of the purified enzyme, which is assumed to be 1:1. A further analysis by sucrose gradient centrifugation revealed that under physiological salt conditions casein kinase 2 from nuclease extracts formed large aggregates (higher than 300 kDa) which were disrupted at 400 mM KCl. At the latter KCl concentration CK-2 activity was localized at a position corresponding to a M(r) of 230-250 kDa, which is still higher than the typical tetrameric form of the enzyme.
通过用脱氧核糖核酸酶I加核糖核酸酶A消化，酪蛋白激酶2从大鼠肝细胞细胞核中释放出来。这种处理还释放出了三种主要底物，分子量分别为50 kDa、40 - 42 kDa和37 kDa。酪蛋白激酶2及其底物也可分别用脱氧核糖核酸酶或核糖核酸酶提取。然而，在脱氧核糖核酸酶提取物中，只有37 kDa的蛋白质能被酪蛋白激酶2磷酸化，而在核糖核酸酶提取物中，所有三种底物都能被磷酸化。当脱氧核糖核酸酶提取物随后用核糖核酸酶处理时，40 - 42 kDa的底物随后被磷酸化，这表明它们与RNA的相互作用阻止了它们被酪蛋白激酶2磷酸化。核酸酶提取物中存在的酪蛋白激酶2的B:α亚基比例高于纯化酶的比例，纯化酶的比例假定为1:1。通过蔗糖梯度离心进一步分析发现，在生理盐条件下，核酸酶提取物中的酪蛋白激酶2形成大聚集体（高于300 kDa），在400 mM KCl时被破坏。在后者的KCl浓度下，CK - 2活性位于对应于230 - 250 kDa分子量的位置，这仍然高于该酶典型的四聚体形式。
