Display options
Cite
Lee JI, Woo SK, Kim KI, et al. A Method for Assaying Deubiquitinating Enzymes. Biol Proced Online. 1998;1:92-99doi: 10.1251/bpo11.
Lee, J. I., Woo, S. K., Kim, K. I., Park, K. C., Baek, S. H., Yoo, Y. J., & Chung, C. H. (1998). A Method for Assaying Deubiquitinating Enzymes. Biological procedures online, 192-99. https://doi.org/10.1251/bpo11
Lee, Jae Il, et al. "A Method for Assaying Deubiquitinating Enzymes." Biological procedures online vol. 1 (1998): 92-99. doi: https://doi.org/10.1251/bpo11
Lee JI, Woo SK, Kim KI, Park KC, Baek SH, Yoo YJ, Chung CH. A Method for Assaying Deubiquitinating Enzymes. Biol Proced Online. 1998 Jul 20;1:92-99. doi: 10.1251/bpo11. PMID: 12734599; PMCID: PMC140113.
Copy Download .nbib Format: NLM
Share it on

Biol Proced Online. 1998 Jul 20;1:92-99. doi: 10.1251/bpo11.

A Method for Assaying Deubiquitinating Enzymes.

Biological procedures online

Jae Il Lee, Seung Kyoon Woo, Keun Il Kim, Kyung Chan Park, Sung Hee Baek, Yung Joon Yoo, Chin Ha Chung

Affiliations

  1. Department of Molecular Biology and Research Center for Cell Differentiation. College of Natural Sciences, Seoul National University, Seoul 151-742. Korea.Department of Life Science. Kwangju Institute of Science and Technology, Kwangju 506-303. Korea. [email protected]

PMID: 12734599 PMCID: PMC140113 DOI: 10.1251/bpo11

Abstract

A general method for the assay of deubiquitinating enzymes was described in detail using (125)I-labeled ubiquitin-fused alphaNH-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.

References

  1. J Biol Chem. 1985 Jun 25;260(12):7609-13 - PubMed
  2. Nature. 1989 Mar 30;338(6214):394-401 - PubMed
  3. Biochemistry. 1995 Nov 7;34(44):14535-46 - PubMed
  4. Biochem J. 1997 Jul 15;325 ( Pt 2):325-30 - PubMed
  5. J Biol Chem. 1996 Jun 14;271(24):14035-40 - PubMed
  6. FASEB J. 1997 Dec;11(14 ):1245-56 - PubMed
  7. Nature. 1984 Dec 13-19;312(5995):663-6 - PubMed
  8. Biochem Soc Trans. 1992 Aug;20(3):631-7 - PubMed
  9. Anal Biochem. 1987 Nov 1;166(2):368-79 - PubMed
  10. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1829-33 - PubMed
  11. Biochemistry. 1995 Oct 3;34(39):12616-23 - PubMed
  12. Annu Rev Genet. 1996;30:405-39 - PubMed
  13. J Biol Chem. 1997 Oct 10;272(41):25560-5 - PubMed
  14. J Biochem. 1997 Apr;121(4):684-9 - PubMed
  15. Anal Biochem. 1982 Sep 15;125(2):427-32 - PubMed
  16. Annu Rev Biochem. 1992;61:761-807 - PubMed
  17. Biochemistry. 1998 Feb 17;37(7):1868-79 - PubMed
  18. Annu Rev Nutr. 1995;15:161-89 - PubMed
  19. J Cell Biol. 1987 Mar;104(3):537-46 - PubMed
  20. Arch Biochem Biophys. 1997 Nov 1;347(1):78-84 - PubMed
  21. Anal Biochem. 1976 May 7;72:248-54 - PubMed
  22. Annu Rev Cell Biol. 1987;3:1-30 - PubMed
  23. Adv Exp Med Biol. 1996;389:203-8 - PubMed
  24. Annu Rev Genet. 1992;26:179-207 - PubMed
  25. J Biol Chem. 1995 Aug 11;270(32):18766-73 - PubMed
  26. J Biol Chem. 1989 Oct 15;264(29):17078-83 - PubMed
  27. J Cell Biol. 1987 Mar;104(3):547-55 - PubMed

Publication Types