Sato H, Yamauchi T, Fujisawa H
Department of Biochemistry, Asahikawa Medical College, Hokkaido.
J Biochem. 1990 Jun;107(6):802-9. doi: 10.1093/oxfordjournals.jbchem.a123129.
A calmodulin-dependent protein kinase has been purified from rat spleen. The enzyme showed a remarkably similar substrate specificity and kinetic parameters to those of rat brain calmodulin-dependent protein kinase II, and exhibited cross-reactivity to a monoclonal antibody against rat brain calmodulin-dependent protein kinase II, indicating that the enzyme might be a calmodulin-dependent protein kinase II isozyme. The sedimentation coefficient was 13.9S, the Stokes radius was 67 A, and the molecular weight was calculated to be 380,000. The purified enzyme gave five polypeptides bands, corresponding to molecular weights of 51,000, 50,000, 21,000, 20,000, and 18,000, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the purified enzyme with Ca2+, calmodulin, and ATP under phosphorylating conditions induced the phosphorylation of all five polypeptides. When the logarithm of the velocity of the phosphorylation was plotted against the logarithm of the enzyme concentration (van't Hoff plot), slopes of 0.89, 0.94, and 1.1 were obtained for the phosphorylation of the 50/51-kDa doublet, 20/21-kDa doublet, and 18-kDa polypeptide, respectively. These results indicate that the phosphorylation of the five polypeptides is an intramolecular process, and further indicate that all five polypeptides are subunits of this enzyme. Of the five polypeptides, only the 50- and 51-kDa polypeptides bound to [125I]calmodulin, the other polypeptides not binding to it. A number of isozymic forms of calmodulin-dependent protein kinase II so far demonstrated in various tissues are known to be composed of subunits with molecular weights of 50,000 to 60,000 which can bind to calmodulin. Thus a new type of calmodulin-dependent protein kinase II was demonstrated in the present study.
一种钙调蛋白依赖性蛋白激酶已从大鼠脾脏中纯化出来。该酶与大鼠脑钙调蛋白依赖性蛋白激酶II表现出显著相似的底物特异性和动力学参数，并与抗大鼠脑钙调蛋白依赖性蛋白激酶II的单克隆抗体呈现交叉反应，这表明该酶可能是一种钙调蛋白依赖性蛋白激酶II同工酶。沉降系数为13.9S，斯托克斯半径为67 Å，计算得到的分子量为380,000。纯化后的酶在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上呈现出五条多肽带，对应分子量分别为51,000、50,000、21,000、20,000和18,000。在磷酸化条件下，将纯化后的酶与Ca2 +、钙调蛋白和ATP一起孵育会诱导所有五条多肽发生磷酸化。当将磷酸化速度的对数与酶浓度的对数作图（范特霍夫图）时，50/51-kDa双峰、20/21-kDa双峰和18-kDa多肽磷酸化的斜率分别为0.89、0.94和1.1。这些结果表明这五条多肽的磷酸化是一个分子内过程，进一步表明所有五条多肽都是该酶的亚基。在这五条多肽中，只有50-kDa和51-kDa多肽与[125I]钙调蛋白结合，其他多肽不与之结合。迄今为止在各种组织中已证明的许多钙调蛋白依赖性蛋白激酶II同工型已知是由分子量为50,000至60,000且能与钙调蛋白结合的亚基组成。因此，本研究中证明了一种新型的钙调蛋白依赖性蛋白激酶II。
