Renart M F, Sastre L, Sebastián J
Eur J Biochem. 1984 Apr 2;140(1):47-54. doi: 10.1111/j.1432-1033.1984.tb08065.x.
A cyclic-AMP-independent nuclear protein kinase has been purified from Dictyostelium discoideum amoebae. The purification procedure involves chromatography of DEAE-Sephadex, phosphocellulose and heparin-Sepharose. The purified enzyme phosphorylates threonine and serine of acidic proteins as casein and phosvitin. Phosphorylation of casein is stimulated by spermine. The kinase requires Mg2+ and can utilize both ATP and GTP as phosphoryl donors. Heparin is a potent inhibitor of the enzyme, being the protein kinase activity fully inhibited at concentrations of 0.5 micrograms/ml. One polypeptide of molecular mass 38 kDa was the major protein band present in the purified kinase preparation as estimated by NaDodSO4 denaturing polyacrylamide gel electrophoresis. This band belongs to the protein kinase because it is the only one that is observed associated with the protein kinase activity when the enzyme preparation is centrifuged in glycerol gradients. The 38-kDa polypeptide is also the major product of autophosphorylation of the enzyme preparation. The enzymatic properties allow to classify the enzyme as a type-II casein kinase. However, its structural properties are different from the mammalian type-II casein kinases and make the D. discoideum enzyme more similar to the plants type-II casein kinases.
已从盘基网柄菌变形虫中纯化出一种不依赖环磷酸腺苷的核蛋白激酶。纯化过程包括用二乙氨基乙基葡聚糖、磷酸纤维素和肝素琼脂糖进行层析。纯化后的酶可将酸性蛋白质（如酪蛋白和卵黄高磷蛋白）的苏氨酸和丝氨酸磷酸化。精胺可刺激酪蛋白的磷酸化。该激酶需要Mg2+，并且既可以利用ATP也可以利用GTP作为磷酸供体。肝素是该酶的有效抑制剂，在浓度为0.5微克/毫升时可完全抑制蛋白激酶活性。通过十二烷基硫酸钠变性聚丙烯酰胺凝胶电泳估计，纯化的激酶制剂中存在的主要蛋白条带是一条分子量为38 kDa的多肽。这条带属于蛋白激酶，因为当酶制剂在甘油梯度中离心时，它是唯一与蛋白激酶活性相关的条带。38 kDa的多肽也是酶制剂自身磷酸化的主要产物。该酶的特性使其可归类为II型酪蛋白激酶。然而，其结构特性与哺乳动物的II型酪蛋白激酶不同，使得盘基网柄菌的这种酶与植物的II型酪蛋白激酶更为相似。
