title: Strategies for assaying deubiquitinating enzymes.
abstract: A general method for assaying deubiquitinating enzymes (DUBs) has been developed. This new method employs an indirect enzyme assay for determining the activity of DUBs using a linear fusion of polyHis-glutathione-S-transferase-ubiquitin-ecotin (His-GST-Ub-ecotin) as a substrate. Because ecotin, a trypsin inhibitor protein from Escherichia coli, is heat stable, the activity of DUBs can be assayed indirectly by determining the ability of ecotin to inhibit trypsin after incubation of any DUB with His-GST-Ub-ecotin followed by heating at 100 degrees. In the substrate construction, His-GST fusion to Ub was used for facilitation of the substrate purification as well as for assisting the heat precipitation of His-GST-Ub and uncleaved His-GST-Ub-ecotin, as Ub itself is also heat stable. This method can also be used for assaying the proteases that process Ub-like proteins (Ubls) using the substrates, in which Ub is replaced by Ubls.
authors.affiliation: School of Biological Sciences, Seoul National University, Seoul 151-742, Korea.
authors.forename: Sung Hwan
authors.initials: SH
authors.lastname: Kang
authors.forename: Jung Jun
authors.initials: JJ
authors.lastname: Park
authors.forename: Sung Soo
authors.initials: SS
authors.lastname: Chung
authors.forename: Ok Sun
authors.initials: OS
authors.lastname: Bang
authors.forename: Chin Ha
authors.initials: CH
authors.lastname: Chung
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin; D014361:Trypsin Inhibitors; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
chemical_list: C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors; D025801:Ubiquitin; C033223:polyhistidine; D006639:Histidine; D010450:Endopeptidases; D014357:Trypsin; C091939:ubiquitin isopeptidase; D005978:Glutathione