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  • Open Access

A method for assaying deubiquitinating enzymes

  • 1,
  • 1,
  • 1,
  • 1,
  • 1,
  • 2, 1 and
  • 1Email author
Biological Procedures Online1:110092

https://doi.org/10.1251/bpo11

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.

Keywords

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

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