Majetschak M, Laub M, Klocke C, Steppuhn J A, Jennissen H P
Institut für Physiologische Chemie, AG Biochemische Endokrinologie, Universität-GHS-Essen, Germany.
Eur J Biochem. 1998 Jul 15;255(2):492-500. doi: 10.1046/j.1432-1327.1998.2550492.x.
Ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21), which has been detected in all tissues so far examined, catalyzes the Ca2+-dependent reversible synthesis of ubiquityl-calmodulin which is not directed to degradation by the ATP-dependent 26-S protease [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191]. As has been shown in the preceding paper in this journal, the uCaM synthetase holosystem can be separated into two essential protein components: uCaM Syn-F1, a ubiquitin-binding protein belonging to the ubiquitin-activating enzyme family (E1) and uCaM Syn-F2 which bestows the reaction specificity leading to the covalent modification of calmodulin with ubiquitin. UCaM Syn-F2, which binds to calmodulin-Sepharose in a Ca2+-dependent manner, has been purified over 3500-fold in seven steps from rabbit reticulocytes and has a native molecular mass of approximately 620 kDa. It binds calmodulin with a Km of 5 microM and to uCaM Syn-F1, i.e. ubiquitin-activating enzyme (E1), with a Km of 3 nM. The maximal specific activity obtained in enriched uCaM Syn-F2 is 6-8 pkat/mg. The pH optimum of uCaM synthetase lies at pH 8.5. In kinetic experiments the Km values for 125I-ubiquitin and ATP/Mg2+ were determined to be 8 microM and 16 nM, respectively, for the uCaM synthetase holosystem. The existence of a third separable protein component of uCaM synthetase, as is the case in E1, E2, E3 systems, is very unlikely since affinity chromatography on calmodulin-Sepharose, two ion-exchange chromatography steps and finally a gel-filtration step failed to indicate any additional protein component essential for synthetase activity. We therefore propose a two-component model for uCaM synthetase. This model is also supported by simple hyperbolic velocity curves in kinetic experiments based on the variation of these two components. The data suggests that uCaM Syn-F2 is neither an E2 nor an E3 but evidently combines the properties of both, making the Ca2+-dependent uCaM synthetase the member of a group of two-component ubiquitin ligase systems.
泛素 - 钙调蛋白连接酶（uCaM合成酶：EC 6.3.2.21），在迄今检测的所有组织中均有发现，它催化钙调蛋白泛素化的钙离子依赖性可逆合成，这种合成并非由ATP依赖性26 - S蛋白酶介导的降解过程[劳布，M. & 詹尼森，H. P.（1997年）《生物化学与生物物理学学报》1357，173 - 191]。正如本刊上一篇论文所表明的，uCaM合成酶全系统可分离为两个必需的蛋白质组分：uCaM Syn - F1，一种属于泛素激活酶家族（E1）的泛素结合蛋白；以及uCaM Syn - F2，它赋予反应特异性，导致钙调蛋白与泛素发生共价修饰。以钙离子依赖性方式与钙调蛋白琼脂糖结合的uCaM Syn - F2，已从兔网织红细胞中经七步纯化了3500多倍，其天然分子量约为620 kDa。它与钙调蛋白结合的Km值为5 μM，与uCaM Syn - F1，即泛素激活酶（E1）结合的Km值为3 nM。富集的uCaM Syn - F2所获得的最大比活性为6 - 8 pkat/mg。uCaM合成酶的最适pH值为8.5。在动力学实验中，对于uCaM合成酶全系统，125I - 泛素和ATP/Mg2 + 的Km值分别测定为8 μM和16 nM。与E1、E2、E3系统的情况一样，uCaM合成酶不太可能存在第三种可分离的蛋白质组分，因为在钙调蛋白琼脂糖上的亲和层析、两步离子交换层析以及最后的凝胶过滤步骤均未表明存在任何对合成酶活性必不可少的其他蛋白质组分。因此，我们提出uCaM合成酶的双组分模型。基于这两个组分的变化进行的动力学实验中简单的双曲线速度曲线也支持了该模型。数据表明，uCaM Syn - F2既不是E2也不是E3，但显然兼具两者的特性，使得钙离子依赖性uCaM合成酶成为双组分泛素连接酶系统中的一员。
