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  • Title: Multiple ubiquitin C-terminal hydrolases from chick skeletal muscle.
    Author: Woo SK, Lee JI, Park IK, Yoo YJ, Cho CM, Kang MS, Ha DB, Tanaka K, Chung CH.
    Journal: J Biol Chem; 1995 Aug 11; 270(32):18766-73. PubMed ID: 7642526.
    Abstract:
    A new method for assaying ubiquitin C-terminal hydrolases was developed using a 125I-labeled ubiquitin-alpha NH-MHISPPEPESEEEEEHYC was substrate. Since the peptide portion was almost exclusively radiolabeled, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid-soluble products. Using this assay protocol, we identified at least 10 ubiquitin C-terminal hydrolase activities from the extract of chick skeletal muscle, which were tentatively named UCHs 1 through 10. Of these, UCH-6 was purified to apparent homogeneity. Purified UCH-6 behaved as a dimer of 27-kDa subunits. The apparent molecular masses of the other partially purified UCHs ranged from 35 to 810 kDa as determined under a non-denaturing condition. Muscle UCHs, except UCH-1, were activated dramatically by poly-L-Lys but with an unknown mechanism. All of the UCHs were sensitive to inhibition by sulfhydryl-blocking agents such as iodoacetamide. In addition, all of the UCHs were capable of releasing free ubiquitin from a ubiquitin-alpha NH-carboxyl extension protein of 80 amino acids and from ubiquitin-alpha NH-dihydrofolate reductase. Five of the enzymes, UCHs 1 through 5, were also capable of generating free ubiquitin from poly-His-tagged diubiquitin. In addition, UCH-1 and UCH-7 could remove ubiquitin that had been ligated covalently by an isopeptide linkage to a ubiquitin (RGA)-alpha NH-peptide, the peptide portion of which consists of the 20 amino acids of the calmodulin binding domain of myosin light chain kinase. These results suggest that the 10 UCH activities isolated from chick skeletal muscle appear to be distinct from each other at least in their chromatographic behavior, size, and substrate specificity.
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