THE UBIQUITYL-CALMODULIN SYNTHETASE SYSTEM FROM RABBIT RETICULOCYTES - ISOLATION OF THE UBIQUITIN-BINDING FIRST COMPONENT, A UBIQUITIN-ACTIVATING ENZYME

Ubiquitin is often implicated as a specific tag fur protein degradatio n via the ubiquitin system although only a limited number of physiolog ical proteins have been shown to be degraded in their native tissues v ia this pathway in vivo. Ubiquitin may also, however, have other funct ions of a regulatory nature (non-catabolic ubiquitylation). The ubiqui tylation of calmodulin appears to fall into this category. Ubiquitin i s linked to free calmodulin in the presence of the second messenger Ca 2+ by the enzyme ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3. 2.21) and there is no evidence that this step is followed by degradati on of calmodulin via the ATP-dependent 26-S protease. Due to a lack of natural substrates and sufficient tissue material, only a few compone nts of the ubiquitin system have been obtained in truly homogenous for m from reticulocytes. We therefore decided to attempt this for the cal modulin ligase. The enzymic components of the uCaM synthetase system c opurified over several steps and could be highly enriched by a novel s ample 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 co mponents: a ubiquitin-Sepharose binding fraction (uCaM Syn-F1) and a c almodulin-Sepharose binding fraction (uCaM Syn-F2), The full activity of uCaM synthetase can be reconstituted when these two fractions are r eunited, uCaM Syn-F1 could then be separated from all other enzymes of ubiquitin metabolism and, employing the second component with the nat ural substrate calmodulin, could be purified over 3500-fold to homogen eity. The ability to catalyze its own thiol labile ubiquitylation iden tified it as a member of the ubiquitin-activating enzyme family (E1). The homogenous preparation contained a single protein of molecular mas s 213 +/- 21 kDa (mean +/- SEM) as determined by gel filtration, The m olecular mass of the monomer was determined by electrospray ion mass s pectrometry to 112 140 +/- 37 Da (mean +/- SD). N-terminal sequence an alysis (20 amino acids) led to a single N-terminal peptide beginning a t residue 57 of the known rabbit cDNA sequence. No ragged N-terminus w as detected, as would be expected by the action of an aminopeptidase o r other peptidases of low specificity. The monomer molecular mass calc ulated from the cDNA sequence (Arg57-Arg1058) is 111975 Da, characteri zing this enzyme from reticulocytes as a homodimer of 224 kDa.