کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1929994 | 1536784 | 2011 | 4 صفحه PDF | دانلود رایگان |
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.
Journal: Biochemical and Biophysical Research Communications - Volume 416, Issues 1–2, 9 December 2011, Pages 76–79