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A Method for Assaying Deubiquitinating Enzymes.

Jae Il Lee1, Seung Kyoon Woo, Keun Il Kim

  • 1Department 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. chchung@plaza.snu.ac.kr

Biological Procedures Online
|May 8, 2003
PubMed
Summary
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A novel assay using radioiodinated ubiquitin-fused protein (Ub-PESTc) allows for direct measurement of deubiquitinating enzyme activity. This method facilitated the purification and comparison of six deubiquitinating enzymes from chick skeletal muscle and yeast.

Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Deubiquitinating enzymes (DUBs) play crucial roles in cellular processes by removing ubiquitin.
  • Assaying DUB activity is essential for their purification and functional characterization.
  • Existing methods may lack sensitivity or require complex procedures.

Purpose of the Study:

  • To develop a general and sensitive assay for deubiquitinating enzymes.
  • To utilize the assay for the purification and comparative analysis of DUBs.
  • To establish a method suitable for identifying eukaryotic DUBs expressed in prokaryotic systems.

Main Methods:

  • Development of a radioiodinated substrate: (125)I-labeled ubiquitin-fused alphaNH-MHISPPEPESEEEEEHYC (Ub-PESTc).
  • Mild radioiodination of a specific tyrosine residue in the PESTc portion of Ub-PESTc using IODO-BEADS.

Related Experiment Videos

  • Assay based on measuring released radioactivity into acid-soluble products, indicating enzyme activity.

    • Main Results:

    • The assay allowed direct measurement of DUB activity by quantifying released radioactivity.
    • Six deubiquitinating enzymes were successfully purified from chick skeletal muscle and yeast.
    • Specific activities of the purified DUBs were compared using the developed assay.
    • Bacterial extracts (E. coli) showed minimal to no activity against the Ub-PESTc substrate.

    Conclusions:

    • The developed assay provides a simple and effective method for quantifying deubiquitinating enzyme activity.
    • This protocol is valuable for the purification and characterization of eukaryotic DUBs.
    • The assay is suitable for identifying and purifying eukaryotic DUBs cloned and expressed in E. coli.