Gregori L, Marriott D, Putkey J A, Means A R, Chau V
J Biol Chem. 1987 Feb 25;262(6):2562-7.
Calmodulin purified from bacteria which express a cloned chicken calmodulin gene can be selectively conjugated with ubiquitin, using enzymes present in reticulocyte extracts. Analyses of peptide products generated from limited proteolytic digestion of the calmodulin conjugate containing a single ubiquitin indicate that lysine 115 on calmodulin is the site of linkage. This linkage site is identical to that previously reported for calmodulin purified from Dictyostelium discoideum. Substrate-dependent ATP hydrolysis by a partially purified ubiquitin conjugation enzyme system from reticulocyte extracts was used to determine the enzyme affinity to calmodulin. Km values of 7 and 9 microM were determined for dictyostelium and the bacterially expressed calmodulin, respectively. The bacterially expressed calmodulin, unlike the Dictyostelium protein, can also form conjugates containing a 2-5 molar ratio of ubiquitin but at a slower rate than that observed for conjugation at lysine 115. Results from these studies further support our hypothesis that the post-translational methylation of lysine 115 found in most forms of calmodulin serves the important function of protecting calmodulin from ubiquitination and from degradation by the cytoplasmic ubiquitin-dependent proteolytic pathway. The capability of the bacterially expressed calmodulin to form conjugates with a high molar ratio of ubiquitin suggests that the post-translational acetylation of the N terminus of calmodulin may serve a similar function.
从表达克隆的鸡钙调蛋白基因的细菌中纯化得到的钙调蛋白，可利用网织红细胞提取物中的酶与泛素选择性结合。对含有单个泛素的钙调蛋白结合物进行有限蛋白酶消化产生的肽产物分析表明，钙调蛋白上的赖氨酸115是连接位点。该连接位点与先前报道的从盘基网柄菌中纯化得到的钙调蛋白的连接位点相同。利用网织红细胞提取物中部分纯化的泛素结合酶系统进行的底物依赖性ATP水解，来确定该酶对钙调蛋白的亲和力。盘基网柄菌和细菌表达的钙调蛋白的Km值分别测定为7和9微摩尔。与盘基网柄菌蛋白不同，细菌表达的钙调蛋白也能形成含有2 - 5摩尔比泛素的结合物，但比在赖氨酸115处结合的速率要慢。这些研究结果进一步支持了我们的假设，即大多数形式的钙调蛋白中发现的赖氨酸115的翻译后甲基化起到了保护钙调蛋白不被泛素化以及不被细胞质中依赖泛素的蛋白水解途径降解的重要作用。细菌表达的钙调蛋白形成高摩尔比泛素结合物的能力表明，钙调蛋白N端的翻译后乙酰化可能起到类似的作用。
