Majetschak M, Laub M, Meyer H E, Jennissen H P
Institut für Physiologische Chemie, AG Biochemische Endokrinologie, Universität-GHS-Essen, Germany.
Eur J Biochem. 1998 Jul 15;255(2):482-91. doi: 10.1046/j.1432-1327.1998.2550482.x.
Ubiquitin is often implicated as a specific tag for protein degradation via the ubiquitin system although only a limited number of physiological proteins have been shown to be degraded in their native tissues via this pathway in vivo. Ubiquitin may also, however, have other functions of a regulatory nature (non-catabolic ubiquitylation). The ubiquitylation of calmodulin appears to fall into this category. Ubiquitin is linked to free calmodulin in the presence of the second messenger Ca2+ by the enzyme ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21) and there is no evidence that this step is followed by degradation of calmodulin via the ATP-dependent 26-S protease. Due to a lack of natural substrates and sufficient tissue material, only a few components of the ubiquitin system have been obtained in truly homogeneous form from reticulocytes. We therefore decided to attempt this for the calmodulin ligase. The enzymic components of the uCaM synthetase system copurified over several steps and could be highly enriched by a novel sample displacement technique on an ion-exchange resin. A fractionation of the synthetase components by affinity chromatography on ubiquitin-Sepharose and calmodulin-Sepharose yielded two essentially inactive components: a ubiquitin-Sepharose binding fraction (uCaM Syn-F1) and a calmodulin-Sepharose binding fraction (uCaM Syn-F2). The full activity of uCaM synthetase can be reconstituted when these two fractions are reunited. uCaM Syn-F1 could then be separated from all other enzymes of ubiquitin metabolism and, employing the second component with the natural substrate calmodulin, could be purified over 3500-fold to homogeneity. The ability to catalyze its own thiol labile ubiquitylation identified it as a member of the ubiquitin-activating enzyme family (E1). The homogeneous preparation contained a single protein of molecular mass 213 +/- 21 kDa (mean +/- SEM) as determined by gel filtration. The molecular mass of the monomer was determined by electrospray ion mass spectrometry to 112,140 +/- 47 Da (mean +/- SD). N-terminal sequence analysis (20 amino acids) led to a single N-terminal peptide beginning at residue 57 of the known rabbit cDNA sequence. No ragged N-terminus was detected, as would be expected by the action of an aminopeptidase or other peptidases of low specificity. The monomer molecular mass calculated from the cDNA sequence (Arg57-Arg1058) is 111,975 Da, characterizing this enzyme from reticulocytes as a homodimer of 224 kDa.
泛素通常被认为是通过泛素系统进行蛋白质降解的特定标签，尽管在体内只有有限数量的生理蛋白质已被证明在其天然组织中通过该途径降解。然而，泛素也可能具有其他调节性质的功能（非分解代谢泛素化）。钙调蛋白的泛素化似乎属于这一类别。在第二信使Ca2+存在的情况下，泛素通过泛素 - 钙调蛋白连接酶（uCaM合成酶：EC 6.3.2.21）与游离钙调蛋白相连，并且没有证据表明这一步骤之后会通过依赖ATP的26 - S蛋白酶降解钙调蛋白。由于缺乏天然底物和足够的组织材料，仅从网织红细胞中以真正均一的形式获得了泛素系统的少数成分。因此，我们决定尝试对钙调蛋白连接酶进行此操作。uCaM合成酶系统的酶成分经过几步共纯化，并可通过离子交换树脂上的新型样品置换技术高度富集。通过泛素 - 琼脂糖和钙调蛋白 - 琼脂糖亲和色谱对合成酶成分进行分级分离，得到两个基本上无活性的成分：一个泛素 - 琼脂糖结合部分（uCaM Syn - F1）和一个钙调蛋白 - 琼脂糖结合部分（uCaM Syn - F2）。当这两个部分重新结合时，uCaM合成酶的全部活性可以恢复。然后可以将uCaM Syn - F1与泛素代谢的所有其他酶分离，并使用含有天然底物钙调蛋白的第二成分，将其纯化至超过3500倍的纯度。催化其自身硫醇不稳定泛素化的能力将其鉴定为泛素激活酶家族（E1）的成员。通过凝胶过滤测定，均一制剂包含一种分子量为213±21 kDa（平均值±标准误）的单一蛋白质。通过电喷雾离子质谱法测定单体的分子量为112,140±47 Da（平均值±标准差）。N端序列分析（20个氨基酸）导致一个单一的N端肽段，起始于已知兔cDNA序列的第残基57。未检测到参差不齐的N端，这是预期的氨基肽酶或其他低特异性肽酶作用的结果。根据cDNA序列（Arg57 - Arg1058）计算的单体分子量为111,975 Da，将网织红细胞中的这种酶表征为224 kDa的同二聚体。
