| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1929994 | Biochemical and Biophysical Research Communications | 2011 | 4 Pages |
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 (UbF45W-Xaa) and di-ubiquitin chains (UbF45W-diUb). After removal of the intact substrate by Ni2+-NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the UbF45W 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.
► A deubiquitinating enzyme has its unique substrate specificity for deubiquitination. ► We have established an activity assay for ubiquitin C-terminal hydrolases. ► This assay can be applicable to other deubiquitinating enzymes.
