Ku K Y, Butcher F R
Biochim Biophys Acta. 1980 Aug 1;631(1):70-8. doi: 10.1016/0304-4165(80)90054-9.
Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca2+ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca2+ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatrography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.
偶联到琼脂糖凝胶上的钙调蛋白提供了一种快速且灵敏的方法，用于从大鼠腮腺中分离出一种受钙调蛋白 - Ca2+ 复合物刺激的环核苷酸磷酸二酯酶活性。初步实验表明，在大鼠腮腺的粗提物中，或在大鼠腮腺磷酸二酯酶经二乙氨基乙基纤维素（DEAE - 纤维素）部分纯化后，均无法证明存在对钙调蛋白和 Ca2+ 敏感的磷酸二酯酶活性。然而，如果首先通过在 DEAE - 纤维素上进行分批离子交换色谱法，然后用偶联到琼脂糖凝胶上的钙调蛋白进行亲和色谱法对提取物进行纯化，就能够很容易地证明存在受钙调蛋白调节的环核苷酸磷酸二酯酶活性。分批离子交换色谱步骤去除了腮腺中大部分游离的钙调蛋白，这些游离钙调蛋白会与偶联到琼脂糖凝胶上的钙调蛋白竞争受钙调蛋白调节的蛋白质。因此，通过在 DEAE - 纤维素上进行初始色谱步骤以将磷酸二酯酶活性与钙调蛋白分离，在用偶联到琼脂糖凝胶上的钙调蛋白进行亲和色谱后，可以提高对钙调蛋白敏感的磷酸二酯酶的回收率。这种方法对于证明受钙调蛋白调节的其他腮腺蛋白的存在以及对其进行纯化应该是有用的。
