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A method for assaying deubiquitinating enzymes

Biological Procedures Online volume 1, pages 92–99 (1998)Cite this article

Abstract

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.

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Authors and Affiliations

  1. Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, 151-742, Seoul, Korea

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

  2. Department of Life Science, Kwangju Institute of Science and Technology, 506-303, Kwangju, Korea

    Yung Joon Yoo

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  1. Jae Il Lee
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  2. Seung Kyoon Woo
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  3. Keun Il Kim
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  4. Kyung Chan Park
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  5. Sung Hee Baek
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  6. Yung Joon Yoo
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  7. Chin Ha Chung
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Correspondence to Chin Ha Chung.

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Lee, J.I., Woo, S.K., Kim, K.I. et al. A method for assaying deubiquitinating enzymes. Biol Proced Online 1, 92–99 (1998). https://doi.org/10.1251/bpo11

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Biological Procedures Online

ISSN: 1480-9222

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