A Method for Assaying Deubiquitinating Enzymes.
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.
Lee
Jae Il
JI
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. chchung@plaza.snu.ac.kr
Woo
Seung Kyoon
SK
Kim
Keun Il
KI
Park
Kyung Chan
KC
Baek
Sung Hee
SH
Yoo
Yung Joon
YJ
Chung
Chin Ha
CH