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Query: UNIPROT:P62988 (Ubiquitin)
 4,326 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In plants Ca2+ plays a crucial role as second messenger. Thus calmodulin is one of the most important signal transducing molecules for metabolic regulation in plants. Previously we showed that bovine testis calmodulin can be covalently coupled at one site to ubiquitin in a Ca2(+)-dependent manner in the presence of ATP/Mg2+ by ubiquityl-calmodulin synthetase. Since calmodulin from spinach has 13 amino acid sequence differences to bovine calmodulin - two of them in Ca2(+)-binding loops - it was unclear, whether a conjugation of ubiquitin to this molecule would be possible. In this paper it is shown that calmodulin from spinach and a similar calmodulin from the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca2(+)-dependent manner. It is shown that higher molecular mass conjugates containing up to three ubiquitin molecules per calmodulin are obtained. Experiments with methylated ubiquitin demonstrate that, as with vertebrate calmodulins, only one lysine residue is linked to ubiquitin and that the incorporation of additional ubiquitin molecules leads to a polyubiquitin chain.
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PMID:Plant and fungus calmodulins are polyubiquitinated at a single site in a Ca2(+)-dependent manner. 217 31

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.
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PMID:The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the calmodulin-binding second component and enzymatic properties. 971 92