A SENSITIVE METHOD FOR DETECTION OF CALMODULIN-DEPENDENT PROTEIN KINASE-II ACTIVITY IN SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL
A SENSITIVE METHOD FOR DETECTION OF CALMODULIN-DEPENDENT PROTEIN KINASE-II ACTIVITY IN SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL
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DOI：
10.1016/0003-2697(89)90181-4
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发表时间：
1989-11-15
影响因子：
2.9
通讯作者：
FUJISAWA, H
中科院分区：
文献类型：
--
作者：
KAMESHITA, I;FUJISAWA, H
一种检测α蛋白激酶活性的方法。和测试版。本文报道了十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分离钙调蛋白依赖性蛋白激酶II亚基的方法。电泳后，凝胶在6M盐酸胍中浸泡1h，然后在含0.04%吐温40R16h的缓冲液中4℃复性。凝胶中的复性多肽与[γ-32P]ATP孵育，以使包括在聚丙烯酰胺凝胶中的底物或激酶本身的磷酸化。去除未反应的[~(32)P]-ATP后，用放射自显影观察蛋白激酶活性。MR 50,000和60,000两条放射性蛋白条带，与α相对应。和测试版。只有在钙离子和钙调蛋白存在下进行磷酸化时，才能检测到亚基。在不含蛋白质底物的凝胶上可以检测到大约0.05µg的酶。当凝胶中含有微管相关蛋白2时，凝胶中钙调素依赖性蛋白激酶II的检测灵敏度比不含蛋白底物的凝胶高一个数量级以上。
A procedure for detecting protein kinase activities of the .alpha. and .beta. subunits of calmodulin-dependent protein kinase II separated by sodium dodecyl sulfate-poly acrylamide gel electrophoresis is described. After electrophoresis, the gel was immersed in 6 M guanidine HCl for 1 h and then in a buffer containing 0.04% Tween 40 fo r16 h at 4.degree.C for renaturation of the resolved polypeptides. The renatured polypeptides in the gel were incubated with [.gamma.-32P]ATP for phosphorylation of either the substrate included in the polyacrylamide gel or the kinase itself. After removal of the unreacted [.gamma.-32P]-ATP, the protein kinase activities were visualized by autoradiography. Two radioactive protein bands of Mr 50,000 and 60,000 which corresponded to the .alpha. and .beta. subunits, were detected only when the phosphorylation was carried out in the presence of Ca2+ and calmodulin. Approximately 0.05 .mu.g of the enzyme could be detected on a gel containing no protein substrate. When microtubule-associated protein 2 was included in the gel, the sensitivity of the detection of calmodulin-dependent protein kinase II in the gel was more than one order of magnitude higher than that in the gel containing no protein substrate.