화학공학소재연구정보센터
Biochemical and Biophysical Research Communications, Vol.416, No.1-2, 76-79, 2011
A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes
Ubiquitin C-terminal hydrolases (UCHs) are a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin,(Ub) chains or extensions. Here we present a convenient method for characterizing the substrate specificities of various UCHs by fluorescently mutated Ub-fusion proteins (Ub(F45W)-Xaa) and di-ubiquitin chains (Ub(F45W)-diUb). After removal of the intact substrate by Ni(2+)-NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the Ub(F45W) product. The results show that three UCHs, i.e. UCH-L1. UCH-L3 and UCH37/UCH-L5, are distinct in their substrate specificities for the Ub-fusions and diUb chains. This assay method may also be applied to study the enzymatic activities and substrate specificities of other DUBs. (C) 2011 Elsevier Inc. All rights reserved.