Yamamoto T, Iwata S
Department of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Meijo University, Nagoya, Japan.
Jpn J Ophthalmol. 1987;31(4):570-81.
Calmodulin and calmodulin-dependent functional protein play an important role in the maintenance of lens transparency and homeostasis. In the present study, phosphodiesterase, one of the typical calmodulin-dependent functional proteins, was purified from bovine lens by DEAE-cellulose chromatography, calmodulin-Sepharose 4B chromatography and Superose 12 chromatography. Moreover, calmodulin-dependent phosphodiesterase, and independent phosphodiesterase were separated from crude lens extract using DEAE-cellulose column. The calmodulin-dependent phosphodiesterase was purified 4500-fold with a 0.7% yield; it was a dimer formed with two single polypeptides of 59K as the molecular weight. The enzyme had a higher affinity for cyclic GMP than for cyclic AMP, and functioned at calcium ion concentration above 10(-6) M in the incubation mixture. W-7 as calmodulin antagonist indirectly inhibited the enzyme activity and nifedipine as calmodulin-dependent phosphodiesterase antagonist directly inhibited the enzyme activity. These results suggest that an appearance of calmodulin-dependent phosphodiesterase activity depends on the interrelation between the calcium ion and calmodulin in the lens.
钙调蛋白和钙调蛋白依赖性功能蛋白在维持晶状体透明度和体内平衡方面发挥着重要作用。在本研究中，通过DEAE-纤维素色谱法、钙调蛋白-琼脂糖4B色谱法和Superose 12色谱法从牛晶状体中纯化了磷酸二酯酶，它是典型的钙调蛋白依赖性功能蛋白之一。此外，使用DEAE-纤维素柱从晶状体粗提物中分离出钙调蛋白依赖性磷酸二酯酶和非依赖性磷酸二酯酶。钙调蛋白依赖性磷酸二酯酶纯化了4500倍，产率为0.7%；它是由两条分子量为59K的单一多肽形成的二聚体。该酶对环鸟苷酸的亲和力高于对环腺苷酸的亲和力，并且在孵育混合物中钙离子浓度高于10^(-6) M时发挥作用。W-7作为钙调蛋白拮抗剂间接抑制酶活性，硝苯地平作为钙调蛋白依赖性磷酸二酯酶拮抗剂直接抑制酶活性。这些结果表明，钙调蛋白依赖性磷酸二酯酶活性的出现取决于晶状体中钙离子与钙调蛋白之间的相互关系。
