Multiple Ubiquitin C-terminal Hydrolases from Chick Skeletal Muscle (∗)

• Published in: The Journal of biological chemistry 1995-08, Vol.270 (32), p.18766-18773
• Main Authors: Woo, Seung Kyoon, Lee, Jae Il, Park, Il Kyoo, Yoo, Yung Joon, Cho, Choong Myung, Kang, Man-Sik, Ha, Doo Bong, Tanaka, Keiji, Chung, Chin Ha
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; Animals; Chickens; HIDROLISIS; HYDROLYSE; Molecular Sequence Data; Molecular Weight; MUSCLE; Muscle, Skeletal - enzymology; MUSCULOS; PESO MOLECULAR; POIDS MOLECULAIRE; POLIMORFISMO ENZIMATICO; POLLITO; Polylysine - pharmacology; POLYMORPHISME ENZYMATIQUE; POUSSIN; PROTEASAS; PROTEASE; Protease Inhibitors - pharmacology; PROTEINAS; PROTEINE; PROTEOLISIS; PROTEOLYSE; PURIFICACION; PURIFICATION; Substrate Specificity; Thiolester Hydrolases - analysis; Thiolester Hydrolases - isolation & purification; Thiolester Hydrolases - metabolism; Ubiquitin Thiolesterase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.32.18766

A new method for assaying ubiquitin C-terminal hydrolases was developed using a 125I-labeled ubiquitin-αNH-MHISPPEPESEEEEEHYC as 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 nondenaturing 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-αNH-carboxyl extension protein of 80 amino acids and from ubiquitin-αNH-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)-αNH-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.