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 for protein degradation via the ubiquitin system although only a limited number of physiological proteins have been shown to be degraded in their native tissues via this pathway in vivo. Ubiquitin may also, however, have other functions of a regulatory nature (non-catabolic ubiquitylation). The ubiquitylation of calmodulin appears to fall into this category. Ubiquitin is linked to free calmodulin in the presence of the second messenger Ca2+ by the enzyme ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21) and there is no evidence that this step is followed by degradation of calmodulin via the ATP-dependent 26-S protease. Due to a lack of natural substrates and sufficient tissue material, only a few components of the ubiquitin system have been obtained in truly homogeneous form from reticulocytes. We therefore decided to attempt this for the calmodulin ligase. The enzymic components of the uCaM synthetase system copurified over several steps and could be highly enriched by a novel sample 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 components: a ubiquitin-Sepharose binding fraction (uCaM Syn-F1) and a calmodulin-Sepharose binding fraction (uCaM Syn-F2). The full activity of uCaM synthetase can be reconstituted when these two fractions are reunited. uCaM Syn-F1 could then be separated from all other enzymes of ubiquitin metabolism and, employing the second component with the natural substrate calmodulin, could be purified over 3500-fold to homogeneity. The ability to catalyze its own thiol labile ubiquitylation identified it as a member of the ubiquitin-activating enzyme family (E1). The homogeneous preparation contained a single protein of molecular mass 213 +/- 21 kDa (mean +/- SEM) as determined by gel filtration. The molecular mass of the monomer was determined by electrospray ion mass spectrometry to 112,140 +/- 47 Da (mean +/- SD). N-terminal sequence analysis (20 amino acids) led to a single N-terminal peptide beginning at residue 57 of the known rabbit cDNA sequence. No ragged N-terminus was detected, as would be expected by the action of an aminopeptidase or other peptidases of low specificity. The monomer molecular mass calculated from the cDNA sequence (Arg57-Arg1058) is 111,975 Da, characterizing this enzyme from reticulocytes as a homodimer of 224 kDa.
Institut für Physiologische Chemie, AG Biochemische Endokrinologie, Universität-GHS-Essen, Germany.
M
M
Majetschak
M
M
Laub
H E
HE
Meyer
H P
HP
Jennissen
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D010453:Peptide Synthases / Q000097:blood* / Q000737:chemistry* / Q000302:isolation & purification; D046988:Proteasome Endopeptidase Complex; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D014908:Triticum / Q000201:enzymology; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D002147:Calmodulin; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; C059404:ubiquitin-calmodulin ligase; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D044764:Ubiquitin-Activating Enzymes; D010453:Peptide Synthases