Fukunaga K, Yamamoto H, Matsui K, Higashi K, Miyamoto E
J Neurochem. 1982 Dec;39(6):1607-17. doi: 10.1111/j.1471-4159.1982.tb07994.x.
A Ca2+- and calmodulin-dependent protein kinase was purified from rat brain cytosol fraction to apparent homogeneity at approximately 800-fold and with a 5% yield. The purified enzyme had a molecular weight of 640,000 as determined by gel filtration analysis on Sephacryl S-300 and a sedimentation coefficient of 15.3 S by sucrose density gradient centrifugation, and resulted in a single protein band of MW 49,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the native enzyme has a large molecular weight and consists of 11 to 14 identical subunits. The purified enzyme exhibited Km values of 109 and 30 microM for ATP and chicken gizzard myosin light chain, respectively, and Ka values of 12 nM and 1.9 microM for brain calmodulin and Ca2+, respectively. In addition to myosin light chain, myelin basic protein, casein, arginine-rich histone, microtubule protein, and synaptosomal proteins were phosphorylated by the enzyme in a CA2+- and calmodulin-dependent manner. The purified enzyme was phosphorylated without the addition of the catalytic subunits of cyclic AMP-dependent protein kinase. Our findings indicate that there is a multifunctional Ca2+- and calmodulin-dependent protein kinase in the brain and that this enzyme may regulate the reactions of various endogenous proteins.
一种钙和钙调蛋白依赖性蛋白激酶从大鼠脑细胞质部分纯化至表观均一，纯化倍数约为800倍，产率为5%。通过Sephacryl S - 300凝胶过滤分析测定，纯化后的酶分子量为640,000，经蔗糖密度梯度离心法测定沉降系数为15.3 S，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中呈现一条分子量为49,000的单一蛋白带。这些结果表明，天然酶分子量较大，由11至14个相同亚基组成。纯化后的酶对ATP和鸡胃肌球蛋白轻链的Km值分别为109和30 microM，对脑钙调蛋白和Ca2 +的Ka值分别为12 nM和1.9 microM。除肌球蛋白轻链外，髓鞘碱性蛋白、酪蛋白、富含精氨酸的组蛋白、微管蛋白和突触体蛋白也能被该酶以钙和钙调蛋白依赖性方式磷酸化。纯化后的酶在不添加环磷酸腺苷依赖性蛋白激酶催化亚基的情况下即可被磷酸化。我们的研究结果表明，脑中存在一种多功能钙和钙调蛋白依赖性蛋白激酶，该酶可能调节各种内源性蛋白质的反应。
