THE UBIQUITYL-CALMODULIN SYNTHETASE SYSTEM FROM RABBIT RETICULOCYTES - ISOLATION OF THE CALMODULIN-BINDING 2ND 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+-depe ndent reversible synthesis of ubiquityl-calmodulin which is not direct ed to degradation by the ATP-dependent 26-S protease [Laub, M. & Jenni ssen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191], As has been shown in the preceding paper in this journal, the uCaM synthetase holo system can be separated into two essential protein components: uCaM Sy n-F1, a ubiquitin-binding protein belonging to the ubiquitin-activatin g enzyme family (E1) and uCaM Syn-F2 which bestows the reaction specif icity leading to the covalent modification of calmodulin with ubiquiti n, UCaM Syn-F2, which binds to calmodulin - Sepharose in a Ca2+-depend ent manner, has been purified over 3500-fold in seven steps from rabbi t 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 spe cific 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 m u M and 16 nM, respectively, for the uCaM synthetase holosystem. The e xistence of a third separable protein component of uCaM synthetase. as is the case in E1, E2, E3 systems, is very unlikely since affinity ch romatography on calmodulin-Sepharose. two ion-exchange chromatography steps and finally a gel-filtration step failed to indicate any additio nal 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 tha t uCaM Syn-F2 is neither an E2 nor an E3 but evidently combines the pr oper ties of both, making the Ca2+-dependent uCaM synthetase the membe r of a group of two-component ubiquitin ligase systems.