Animal cells and systems

The Korean Society for Integrative Biology (한국통합생물학회)
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A Novel Ubiqutin C-terminal Hydrolase (UCH-9) from Chick Skeletal Muscle: Its Purification and Charaterization

  • U, Seong-Gyun (Department of Molecular Biology and Reseac Center for Cell Differentiation, Seoul National University) ;
  • Baek, Seong-Hui (Department of Molecular Biology and Reseac Center for Cell Differentiation, Seoul National University) ;
  • Sin, Dong-Hun (Department of Molecular Biology and Reseac Center for Cell Differentiation, Seoul National University) ;
  • Kim, Hye-Seon (Biological Sciences, Ajou University) ;
  • Yu, Yeong-Jun (LG Biotech Ltd.) ;
  • Jo, Jung-Myeong (LG Biotech Ltd.) ;
  • Gang, Man-Sik (Department of Molecular Biology and Reseac Center for Cell Differentiation, Seoul National University) ;
  • Jeong, Jin-Ha (Department of Molecular Biology and Reseac Center for Cell Differentiation, Seoul National University)
  • Published : 1997.06.01
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Abstract

We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). In the present studies, one of the enzymes, called UCH-9, was purified by conventional chromatographic procedures using $^{125}l$-labeled ubiquitin-${\alpha}$NH-MHISPPEPESEEEEE HYC (Ub-PESTc) as a substrate. The purified enzyme behaved as a 27-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. It was maximally active at pHs between 7 and 8.5, but showed little or no activity at pH below 6 and above 10. Lice other UCHs, its activity was strongly inhibited by sulfhydryl blocking reagents, such as iodoacetamide, and by Ub-aldehyde. In addition to Ub-PESTc, UCH-9 hydrolyzed Ub-aNH-protein extensions, including Ub-${\alpha}NH$-carboxyl extension protein of 80 amino acids and Ubo-${\alpha}NH$-dihydrofolate reductase. However, this enzyme was not capable of generating free Ub from mono-Ub-${\varepsilon}NH$-protein conjugates and from branched poly-Ub chains that are ligated to proteins through ${\varepsilon}NH$-isopeptide bonds. This enzyme neither could hydrolyze poly-His-tagged di-Ub. These results suggest that UCH-9 may play an important role in production of free Ub and ribosomal proteins from their conjugates.

Keywords

References

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