Hathaway D R, Adelstein R S, Klee C B
J Biol Chem. 1981 Aug 10;256(15):8183-9.
Myosin light chain kinase and a fraction of type II cAMP-dependent protein kinase have been partially purified from bovine brain by affinity chromatography on calmodulin-Sepharose. The myosin kinase was purified approximately 3700-fold and has an estimated molecular weight of 130,000 +/- 10,000 by sodium dodecyl sulfate gel electrophoresis. A fraction of soluble cAMP-dependent protein kinase also bound to calmodulin-Sepharose and was purified 2300-fold. A fraction of this cAMP-dependent protein kinase after purification by glycerol gradient centrifugation was shown to contain the two subunits of calcineurin, a major calmodulin-binding protein in brain, and the two subunits of type II cAMP-dependent protein kinase in a ratio of 1:1:2:2. Its sedimentation coefficient was 8.1 S and 9.0 S when centrifuged in the absence or presence of calmodulin, suggesting the formation of a complex between calmodulin and protein kinase. Our results suggest the possibility that calcineurin may be involved in the interaction between the protein kinase and calmodulin. Furthermore, our studies imply that the regulatory subunit of the cAMP-dependent protein kinase, but not the catalytic subunit, is the site of interaction with calmodulin since the catalytic subunit of protein kinase was partially resolved from the complex by cAMP.
通过钙调蛋白-琼脂糖亲和层析法，已从牛脑中部分纯化了肌球蛋白轻链激酶和一部分II型环磷酸腺苷依赖性蛋白激酶。肌球蛋白激酶经纯化后约提高了3700倍，通过十二烷基硫酸钠凝胶电泳法估计其分子量为130,000±10,000。一部分可溶性环磷酸腺苷依赖性蛋白激酶也与钙调蛋白-琼脂糖结合，并被纯化了2300倍。经甘油梯度离心纯化后的这部分环磷酸腺苷依赖性蛋白激酶显示，其含有脑内主要的钙调蛋白结合蛋白钙调神经磷酸酶的两个亚基，以及II型环磷酸腺苷依赖性蛋白激酶的两个亚基，比例为1:1:2:2。在无钙调蛋白或有钙调蛋白存在的情况下进行离心时，其沉降系数分别为8.1 S和9.0 S，这表明钙调蛋白与蛋白激酶之间形成了复合物。我们的结果提示，钙调神经磷酸酶可能参与了蛋白激酶与钙调蛋白之间的相互作用。此外，我们的研究表明，环磷酸腺苷依赖性蛋白激酶的调节亚基而非催化亚基是与钙调蛋白相互作用的位点，因为蛋白激酶的催化亚基可通过环磷酸腺苷从复合物中部分解离出来。
