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A method for assaying deubiquitinating enzymes

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A general method for the assay of deubiquitinating enzymes was described in detail using 125I-labeled ubiquitin-fused αNH-MHISPPEPESEEEEEHYC (referred to as Ub-PESTc) as a substrate. Since the tyrosine residue in the PESTc portion of the fusion protein was almost exclusively radioiodinated under a mild labeling condition, such as using IODO-BEADS, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid soluble products. Using this assay protocol, we could purify six deubiquitinating enzymes from chick skeletal muscle and yeast and compare their specific activities. Since the extracts of E. coli showed little or no activity against the substrate, the assay protocol should be useful for identification and purification of eukaryotic deubiquitinating enzymes cloned and expressed in the cells.


  1. 1.

    Rechsteiner, M. 1987. Ubiquitin-mediated pathways for intracellular proteolysis. Annu. Rev. Cell Biol. 3, 1–30.

  2. 2.

    Hershko, A., and Ciechanover, A. 1992. The ubiquitin system for protein degradation. Annu. Rev. Biochem. 61, 761–807.

  3. 3.

    Jentsch, S. 1992. The ubiquitin-conjugating system. Annu. Rev. Genet. 26, 179–207.

  4. 4.

    Hochstrasser, M. 1996. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30, 405–439.

  5. 5.

    Wilkinson, K.D. 1995. Roles of ubiquitinylation in proteolysis and cellular regulation. Annu. Rev. Nutr. 15, 161–189.

  6. 6.

    Carlson, N., and Rechsteiner, M. 1987. Microinjection of ubiquitin: Intracellular distribution of and metabolism in HeLa cells maintained under normal physiological conditions. J. Cell Biol. 104, 537–546.

  7. 7.

    Carlson, N., Rogers, S., and Rechsteiner, M. 1987. Microinjection of ubiquitin: Changes in protein degradation in HeLa cells subjected to heat-shock. J. Cell Biol. 104, 547–555.

  8. 8.

    Haas, A.L. 1988. Immunochemical probes of Ub pool dynamics. In Ubiquitin (Rechsteiner, M., ed.) pp. 173–206, Plenum Press, New York.

  9. 9.

    Ozkaynak, E., Finley, D., and Varshavsky, A. 1984. The yeast ubiquitin gene: Head-to-tail repeats encoding a polyubiquitin precursor protein. Nature 312, 663–666.

  10. 10.

    Lund, P.K., Moasts-Staats, B.M., Simmons, J.G., Hoyt, E., D’Ercole, A.J., Martin, F., and Van Wyk, J.J. 1985. Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesized as a precursor. J. Biol. Chem. 260, 7609–7613.

  11. 11.

    Finley, D., Bartel, B., and Varshavsky, A. 1989. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature 338, 394–401.

  12. 12.

    Wilkinson, K.D. 1997. Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J. 11, 1245–1256.

  13. 13.

    Woo, S.K., Lee, J.I., Park, I.K., Yoo, Y.J., Cho, J.M., Tanaka, K., Kang, M.S., Ha, D.B. and Chung, C.H. 1995. Multiple ubiquitin C-terminal hydrolases from chick skeletal muscle. J. Biol. Chem. 270, 18766–18773.

  14. 14.

    Chung, C.H., Woo, S.K., Lee, J.I., Park, I.K., Kang, M.S. and Ha, D.B. 1996. Ubiquitin Cterminal hydrolases in chick skeletal muscle. Adv. Exp. Med. Biol. 384, 203–208.

  15. 15.

    Baek, S.H., Choi, K.S., Yoo, Y.J., Cho, J.M., Baker, R.T., Tanaka, K. and Chung, C.H. 1997. Molecular cloning of a novel ubiquitin-specific protease, UBP41, with isopeptidase activity in chick skeletal muscle. J. Biol. Chem. 272, 25560–25565.

  16. 16.

    Woo, S.K., Baek, S.H., Lee, J.I., Yoo, Y.J., Cho, J.M., Kang, M.S. and Chung, C.H. 1997. Purification and characterization of a new ubiquitin C-terminal hydrolase (UCH-1) with isopeptidase activity from chick skeletal muscle. J. Biochem. 121, 684–689.

  17. 17.

    Baek, S.H., Woo, S.K., Lee, J.I., Yoo, Y.J., Cho, J.M., Kang, M.S., Tanaka, K. and Chung, C.H. 1997. New de-ubiquitinating enzyme, ubiquitin C-terminal hydrolase 8, in chick skeletal muscle. Biochem. J. 325, 325–330.

  18. 18.

    Park, K.C., Woo, S.K., Yoo, Y.J., Wyndham, A.M., Baker, R.T. and Chung, C.H. 1997. Purification and characterization of UBP6, a new ubiquitin specific protease in Saccharomyces cerevisiae. Arch. Biochem. Biophys. 347, 78–84.

  19. 19.

    Stein, R.L., Chen, Z., and Melandri, F. 1995. Kinetic studies of isopeptidase T: Modulation of peptidase activity by ubiquitin. Biochemistry 34, 12616–12623.

  20. 20.

    Miller, H.I., Henzel, W.J., Ridgway, J.B., Kuang, W.J., Chisholm, V., and Liu, C.C. 1989. Cloning and expression of a yeast ubiquitin-protein cleaving activity in E. coli. Bio/Technology 7, 698–704.

  21. 21.

    Yoo, Y., Rote, K., and Rechsteiner, M. 1989. Synthesis of peptides as cloned ubiquitin extensions. J. Biol. Chem. 264, 17078–17083.

  22. 22.

    Markwell, M.A.K. 1982. A new solid-phase reagent to iodinate proteins. Anal. Biochem. 125, 427–435.

  23. 23.

    Yoo, S.J., Seol, J.H., Shin, D.H., Rohrwild, M., Kang, M.S., Tanaka, K., Goldberg, A.L., and Chung, C.H. 1996. Purification and characterization of the heat shock proteins HslV and HslU that forms a new ATP-dependent protease in Escherichia coli. J. Biol. Chem. 271, 14035–14040.

  24. 24.

    Bradford, M.M. A rapid and sensitive method of quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.

  25. 25.

    Schägger, H., and von Jagow, G. 1987. Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379.

  26. 26.

    Hershko, A., and Rose, I.A. 1977. Ubiquitin-aldehyde: a general inhibitor of ubiquitin-protein ligase system. Proc. Natl. Acad. Sci. U.S.A. 84, 1829–1833.

  27. 27.

    Wilkinson, K.D., Tashayev, V.L., O’Connor, L.B., Larsen, C.N., Kasperek, E., and Pickart, C.M. 1995. Metabolism of the polyubiquitin degradation signal: structure, mechanism, and role of isopeptidase T. Biochemistry 34, 14535–14546.

  28. 28.

    Baek, S.H., and Chung, C.H. manuscript in preparation.

  29. 29.

    Dang, L.C., Melandri, F.D., and Stein, R.L. 1998. Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry 37, 1868–1879.

  30. 30.

    Wilkinson, K.D., Deshpande, S., and Larsen, C.N. 1992. Comparisons of neuronal (PGP 9.5) and non-neuronal ubiquitin C-terminal hydrolases. Biochem. Soc. Trans. 20, 631–636.

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Correspondence to Jae Il Lee or Chin Ha Chung.

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  • Assay Protocol
  • Biological Procedure
  • Strain AR13
  • Deubiquitinating Enzyme
  • Biological Procedure Online