Sharma R K, Wang T H, Wirch E, Wang J H
J Biol Chem. 1980 Jun 25;255(12):5916-23.
Calmodulin-dependent cyclic nucleotide phosphodiesterase was purified from bovine brain to apparent homogeneity by a new procedure involving DEAE-cellulose, Affi-Gel blue, calmodulin-Sepharose 4B, and Sephadex G-200 column chromatographies. The enzyme was purified more than 3,000-fold from the brain extracts with greater than 12% yield. The purified phosphodiesterase could be activated 10- to 15-fold by calmodulin and Ca2+ to a specific enzyme activity of more than 300 mumol of cAMP hydrolyzed/min/mg of protein. Molecular weight of the enzyme was determined to be 115,800 by the sedimentation equilibirum method or 124,000 from the sedimentation constant and Stokes radius of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight of 58,000. These results suggested that the calmodulin-dependent phosphodiesterase from bovine brain has a subunit structure of alpha2. Molecular weight of the complex of calmodulin and phosphodiesterase was the complex of calmodulin and phosphodiesterase was also calculated from the sedimentation constant and Stokes radius to be 159,000. Since calmodulin has a molecular weight of about 17,000, the result indicated that the stoichiometry of the complex is calmodulin2 alpha2. The catalytic subunit of cylic AMP-dependent protein kinase was found to catalyze the phosphorylation of the purified phosphodiesterase with the incorporation of 2 mol of phosphate/mol of the enzyme.
通过一种新的方法，利用二乙氨基乙基纤维素（DEAE - 纤维素）、亲和凝胶蓝、钙调蛋白琼脂糖4B和葡聚糖G - 200柱色谱，从牛脑中纯化出钙调蛋白依赖性环核苷酸磷酸二酯酶至表观均一。该酶从脑提取物中纯化了3000多倍，产率大于12%。纯化的磷酸二酯酶可被钙调蛋白和Ca2 +激活10至15倍，达到超过300 μmol cAMP水解/分钟/毫克蛋白质的比酶活性。通过沉降平衡法测定该酶的分子量为115,800，或根据蛋白质的沉降常数和斯托克斯半径计算为124,000。该酶的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳显示一条单一蛋白带，表观分子量为58,000。这些结果表明，来自牛脑的钙调蛋白依赖性磷酸二酯酶具有α2亚基结构。钙调蛋白和磷酸二酯酶复合物的分子量也根据沉降常数和斯托克斯半径计算为159,000。由于钙调蛋白的分子量约为17,000，结果表明该复合物的化学计量比为钙调蛋白2α2。发现环磷酸腺苷依赖性蛋白激酶的催化亚基催化纯化的磷酸二酯酶的磷酸化，每摩尔酶掺入2摩尔磷酸盐。
