The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: Isolation of the calmodulin-binding second component and enzymatic properties

Ubiquitin-calmodulin ligase (uCaM synthetase; EC 6.3,2.2.1), 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 approximate to 620 kDa. It binds calmodulin with a K-m of 5 mu M and to uCaM Syn-F1, i.e. ubiquitin-activating enzyme (E1), with a K-m 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 K-m values for I-125-ubiquitin and ATP/Mg2+ were determined to be 8 mu M 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 proper ties of both, making the Ca2+-dependent uCaM synthetase the member of a group of two-component ubiquitin ligase systems.