Purification and properties of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase.
Purification and properties of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase.
复制标题
牛脑钙调蛋白依赖性环核苷酸磷酸二酯酶的纯化和特性。
DOI：
10.1016/s0021-9258(19)70718-2
复制
发表时间：
1980
期刊：
影响因子：
--
通讯作者：
J. H. Wang
中科院分区：
文献类型：
--
作者：
R. Sharma;T. Wang;E. Wirch;J. H. Wang
用DEAE-纤维素、Affi-Gel blue、CaM-Sepharose 4 B和Sephadex G-200柱层析从牛脑中纯化钙调素依赖性环核苷酸磷酸二酯酶。该酶从脑提取物中纯化超过3，000倍，产率大于12%。纯化的磷酸二酯酶可以激活10- 15倍的钙调素和Ca ~（2+）的比酶活性超过300 μ mol的cAMP水解/分钟/毫克蛋白质。通过沉降平衡法测定酶的分子量为115，800，或根据蛋白质的沉降常数和斯托克斯半径测定为124，000。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的酶显示一个单一的蛋白质带的表观分子量为58000。结果表明，牛脑钙调素依赖性磷酸二酯酶具有α 2亚基结构。钙调素和磷酸二酯酶复合物的分子量也由沉降常数和斯托克斯半径计算为159，000。由于钙调素的分子量约为17，000，因此结果表明复合物的化学计量为钙调素2 α 2。环腺苷酸依赖性蛋白激酶的催化亚基被发现催化磷酸化的纯化的磷酸二酯酶与掺入2摩尔磷酸/摩尔的酶。
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.