Ziegenhagen R, Goldberg M, Rakutt W D, Jennissen H P
Institut für Physiologische Chemie, Universität-GHS-Essen, FRG.
FEBS Lett. 1990 Oct 1;271(1-2):71-5. doi: 10.1016/0014-5793(90)80374-r.
In the presence of Ca2+ and ATP/Mg2+ mammalian calmodulin can be covalently coupled to ubiquitin by ubiquityl calmodulin synthetase (uCaM-synthetase). Three ubiquitin derivatives 125I-CT-ubiquitin (prepared by the chloramine-T method), 125I-BH-ubiquitin (prepared by the Bolton-Hunter method) and methylated forms of ubiquitin were tested with native calmodulin. Alternatively native ubiquitin was tested with the Bolton-Hunter derivative of calmodulin (125I-BH-calmodulin). Up to three molecules of ubiquitin can be incorporated into one molecule of calmodulin. Since both native forms of ubiquitin and calmodulin are good substrates of uCaM-synthetase, ubiquitination is not a result of an altered conformation (i.e. denaturation) of either protein. With 125I-BH-calmodulin it is demonstrated that calmodulin is also present in the higher molecular weight ubiquitin conjugates. If methylated ubiquitin is employed as substrate for uCaM-synthetase only one conjugate corresponding to the mono-ubiquitination product of calmodulin is formed. This demonstrates that only a single lysine residue in calmodulin is conjugated to ubiquitin. All other higher molecular weight ubiquitin-calmodulin conjugates must therefore be composed of one molecule of calmodulin to which an oligo- or poly-ubiquitin chain is linked. Since it can be shown that the mono-ubiquitination product of calmodulin still contains ca. 1 mol trimethyllysine/mol calmodulin, the poly-ubiquitin chain is not linked to lysine 115 of calmodulin. In addition a demethylation of trimethyllysine 115 by enzymes in reticulocyte lysate or the DEAE-enriched enzyme fraction with subsequent ubiquitination at this site of calmodulin can also be excluded.
在钙离子和三磷酸腺苷/镁离子存在的情况下，哺乳动物钙调蛋白可通过泛素化钙调蛋白合成酶（uCaM合成酶）与泛素共价偶联。用三种泛素衍生物125I-CT-泛素（通过氯胺-T法制备）、125I-BH-泛素（通过博尔顿-亨特法制备）以及泛素的甲基化形式对天然钙调蛋白进行了测试。另外，用钙调蛋白的博尔顿-亨特衍生物（125I-BH-钙调蛋白）对天然泛素进行了测试。一个钙调蛋白分子最多可结合三个泛素分子。由于泛素和钙调蛋白的天然形式都是uCaM合成酶的良好底物，因此泛素化不是任何一种蛋白质构象改变（即变性）的结果。用125I-BH-钙调蛋白证明，在较高分子量的泛素缀合物中也存在钙调蛋白。如果使用甲基化泛素作为uCaM合成酶的底物，仅形成一种与钙调蛋白单泛素化产物相对应的缀合物。这表明钙调蛋白中只有一个赖氨酸残基与泛素缀合。因此，所有其他较高分子量的泛素-钙调蛋白缀合物必定由一个与寡聚或多聚泛素链相连的钙调蛋白分子组成。由于可以证明钙调蛋白的单泛素化产物每摩尔钙调蛋白仍含有约1摩尔三甲基赖氨酸，所以多聚泛素链不是与钙调蛋白的赖氨酸115相连。此外，也可以排除网织红细胞裂解液或DEAE富集酶组分中的酶对三甲基赖氨酸115进行去甲基化，随后在钙调蛋白的该位点进行泛素化的情况。
