Isolation and characterization of calmodulin from spinach leaves and in vitro translation mixtures.
Isolation and characterization of calmodulin from spinach leaves and in vitro translation mixtures.
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从菠菜叶和体外翻译混合物中分离和表征钙调蛋白。
DOI：
10.1073/pnas.77.4.1912
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发表时间：
1980
影响因子：
11.1
通讯作者：
D. Watterson
中科院分区：
文献类型：
--
作者：
L. Eldik;A. Grossman;D. Iverson;D. Watterson
钙调素是一种多功能的钙调节蛋白，已从菠菜叶片组织和菠菜叶片信使RNA翻译产物中分离得到。翻译蛋白和菠菜叶蛋白已被部分鉴定，并与脊椎动物钙调蛋白进行了比较。菠菜叶钙调素将定量激活牛脑磷酸二酯酶，并将经历与牛脑钙调蛋白类似的钙依赖的凝胶迁移率变化。在有Ca(2+)存在的情况下，菠菜和脑蛋白质会发生共烧，但在有螯合剂存在的情况下，它们不会。从菠菜叶片组织中分离出一个多腺化的RNA组分，并在小麦胚芽无细胞翻译系统中进行翻译。体外合成的钙调素通过吩噻嗪-琼脂糖偶联物的钙依赖亲和层析分离得到。在聚丙烯酰胺凝胶电泳法中，无论有无钙离子存在，翻译蛋白都与菠菜钙调蛋白共迁移。该翻译蛋白也经历了与菠菜钙调蛋白相同的钙依赖的迁移率变化。对翻译的钙调蛋白进行的氨基酸分析表明，它不包含N(Epsilon)-三甲基赖氨酸，这是一种氨基酸残基，是所有先前研究的钙调蛋白的特征。这些研究表明，N(Epsilon)-三甲基赖氨酸不是钙依赖的钙调蛋白与吩噻嗪相互作用所必需的，并表明吩噻嗪-琼脂糖偶联物在生物和生化研究中作为亲和性吸附剂的潜在用途。
Calmodulin, a multifunctional calcium-modulated protein, has been isolated from spinach leaf tissue and from spinach leaf messenger RNA translation products. The translation protein and the spinach leaf protein have been partially characterized and compared to vertebrate calmodulins. Spinach leaf calmodulin will quantitatively activate bovine brain phosphodiesterase and will undergo a calcium-dependent shift in electrophoretic mobility similar to that of bovine brain calmodulin. In the presence of Ca(2+) the spinach and brain proteins comigrate, but in the presence of chelators they do not. A polyadenylylated RNA fraction has been isolated from spinach leaf tissue and translated in a wheat germ cell-free translation system. The calmodulin synthesized in vitro has been isolated by using calcium-dependent affinity chromatography on phenothiazine-Sepharose conjugates. The translation protein comigrates with spinach calmodulin during polyacrylamide gel electrophoresis whether in the presence or the absence of Ca(2+). The translation protein also undergoes a calcium-dependent mobility shift identical to that of spinach calmodulin. Amino acid analysis of the translation calmodulin indicates that it does not contain N(epsilon)-trimethyllysine, an amino acid residue that is characteristic of all calmodulins previously examined. These studies suggest that N(epsilon)-trimethyllysine is not required for the calcium-dependent interaction of calmodulin with phenothiazines and indicate the potential utility of phenothiazine-Sepharose conjugates as affinity-based adsorbents in biological and biochemical investigations.