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J. Biochem, 1997, Vol. 121, No. 4 684-689
© 1997 Japanese Biochemical Society

research-article

Purification and Characterization of a New Ubiquitin C-Terminal Hydrolase (UCH-1) with Isopeptidase Activity from Chick Skeletal Muscle1

Seung Kyoon Woo*, Sung Hee Baek*, Lee, Jae II*, Yung Joon Yoo{dagger}, Choong Myung Cho{dagger}, Man-Sik Kang* and Chin Ha Chung*,2

*Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University Seoul 151-742, Korea
{dagger}LG Biotech Ltd. Taejeon 305-380, Korea

2E-mail: chchung{at}plaza.snu.ac.kr

We have previously shown that chick muscle extracts contain at least 10 different ubiquitin C-terminal hydrolases (UCHs). In the present studies, one of the enzymes, called UCH-1 was partially purified by conventional chromatographic procedures using 125I-labeled ubiquitin-{alpha}NH-MHISPPEPESEEEEEHYC as a substrate. The purified enzyme behaved as a 36-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consisted of a single polypeptide chain. It was maximally active at pHs between 8 and 9, but showed little or no activity at pH below 6 and above 11. Like other UCHs, its activity was strongly inhibited by sulfhydryl blocking reagents, such as iodoacetamide, and by ubiquitin-aldehyde. In addition to Ub-PESTc, UCH-1 hydrolyzed ubiquitin-{alpha}NH-protein extensions, including ubiquitin-{alpha}NH-carboxyl extension protein of 80 amino acids, ubiquitin-{alpha}NH-dihydrofolate reductase, and poly-His-tagged di-ubiquitin. This enzyme was also capable of generating free ubiquitin from mono-ubiquitin-{varepsilon}NH-protein conjugates and from branched poly-ubiquitin chains that are ligated toproteins through {varepsilon}NH-isopeptide bonds. These results suggest that UCH-1 may play an important role in the generation of free ubiquitin from ubiquitin-ribosomal protein fusions and linear polyubiquitin, as well as in recycling of Ub molecules after degradation of poly-ubiquitinated protein conjugates by the 26S proteasome.

1This work was supported by grants from Korea Science and Engineering Foundation through the Research Center for Cell Differentiation, The Han Project, and The Ministry of Education of Korea.


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