Kuret J, Schulman H
Biochemistry. 1984 Nov 6;23(23):5495-504. doi: 10.1021/bi00318a018.
A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.
通过使用酪蛋白作为底物，已从大鼠脑中纯化出一种可溶性的钙/钙调蛋白依赖性蛋白激酶，纯度接近均一。该酶通过羟基磷灰石吸附色谱、磷酸纤维素离子交换色谱、琼脂糖6B凝胶过滤、使用钙调蛋白-琼脂糖4B的亲和色谱以及硫酸铵沉淀进行纯化。在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶上，纯化后的酶由三条蛋白带组成：一条51000道尔顿的单一多肽和一条60000道尔顿的双峰。通过凝胶过滤测量斯托克斯半径（81.3±3.7 Å）以及通过蔗糖密度沉降测量沉降系数（13.7±0.7 S），用于计算天然分子量为460000±29000道尔顿。该激酶使51000道尔顿的多肽和60000道尔顿的双峰自身磷酸化，导致在SDS凝胶中的迁移率降低。对自身磷酸化酶进行部分蛋白水解产生的磷酸肽进行比较，发现亚基之间有很大的相似性。然而，这些模式表明51000道尔顿的亚基不是60000道尔顿双峰的蛋白水解片段。当在饱和酪蛋白浓度下测量时，纯化的钙/钙调蛋白依赖性酪蛋白激酶活性依赖于钙离子、钙调蛋白以及ATP·Mg2+或ATP·Mn2+。在镁离子存在且钙调蛋白浓度饱和的情况下，钴离子、锰离子和镧离子可以替代钙离子。除了酪蛋白外，纯化后的酶还表现出广泛的底物特异性，这表明它可能是一种“通用”蛋白激酶，有可能介导大脑以及可能其他组织中的许多过程。
