从心肌中分离出一种缺乏5.3-S钙调蛋白的3':5'-环核苷酸磷酸二酯酶。

Isolation of a 5.3-S calmodulin-deficient 3':5'-cyclic nucleotide phosphodiesterase from cardiac muscle.

Mohindru A, Rhoads A R

Biochem J. 1982 Aug 1;205(2):427-35. doi: 10.1042/bj2050427.

Abstract

In the presence of Ca2+, a 5.3-S 3':5'-cyclic nucleotide phosphodiesterase (EC 3.1.4.17) from bovine ventricle was isolated and purified by (NH4)2SO4 precipitation and DEAE-cellulose and Affi-Gel Blue chromatography. The enzyme activity was enriched 800-fold by these procedures. 2. Sucrose-density gradient centrifugation, gel filtration and non-denaturing polyacrylamide-gel electrophoresis resolved a single enzyme species with an Mr of 89 000. 3. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the purified enzyme demonstrated a prominent protein band at Mr 59000 and a minor band of Mr 28000. Calmodulin was not detected. 4. The hydrolysis of micromolar concentrations of 3':5'-cyclic guanosine monophosphate (cyclic GMP) but not 3':5'-cyclic adenosine monophosphate (cyclic AMP) was stimulated by calmodulin. 5. Anomalous biphasic kinetics plots were observed for both the catalysis of cyclic AMP and cyclic GMP hydrolysis. Kinetic plots became linear in the presence of calmodulin. 6. After several months of storage at -20 degrees C, the 5.3-S enzyme was transformed into a 6.2-S cyclic GMP-specific enzyme and a 4.4-S non-specific form.

摘要

在钙离子存在的情况下，通过硫酸铵沉淀、DEAE-纤维素和Affi-Gel Blue层析法从牛心室中分离并纯化出一种5.3-S 3':5'-环核苷酸磷酸二酯酶（EC 3.1.4.17）。通过这些步骤，酶活性提高了800倍。2. 蔗糖密度梯度离心、凝胶过滤和非变性聚丙烯酰胺凝胶电泳分离出一种单一的酶，其相对分子质量为89000。3. 纯化酶的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳显示在相对分子质量59000处有一条明显的蛋白带，在相对分子质量28000处有一条较弱的带。未检测到钙调蛋白。4. 钙调蛋白刺激微摩尔浓度的3':5'-环鸟苷单磷酸（环鸟苷酸）的水解，但不刺激3':5'-环腺苷单磷酸（环腺苷酸）的水解。5. 观察到环腺苷酸和环鸟苷酸水解催化的异常双相动力学曲线。在钙调蛋白存在下，动力学曲线变为线性。6. 在-20℃储存几个月后，5.3-S酶转变为一种6.2-S环鸟苷酸特异性酶和一种4.4-S非特异性形式。