Ub-AMC
Ubiquitin-AMC is a fluorogenic substrate for a wide range of deubiquitinating enzymes (DUBs), including ubiquitin C-terminal hydrolases (UCHs) and ubiquitin specific proteases (USPs). It is a particularly useful reagent for the study of deubiquitinating activity where detection sensitivity or continuous monitoring of activity is essential.[1]
Background[edit]
Ubiquitin-AMC is prepared by the C-terminal derivatization of ubiquitin with 7-amino-4-methylcoumarin and has been shown to be a useful and sensitive fluorogenic substrate for wide range of deubiquitinylating enzymes (DUBs), including ubiquitin C-terminal hydrolases (UCHs) and ubiquitin specific proteases (USPs).
Ubiquitin-AMC has been shown to be a sensitive substrate for UCH-L3 (Km = 0.039µM) and for Isopeptidase-T (Km = 0.17-1.4µM),[2] and is particularly useful for studying deubiquitinylating activity where detection sensitivity or continuous monitoring of activity is essential.[3]
Typical assay set-up: Assay substrate concentration: 0.01-1.0µM. Enzyme concentrations, UCH-L3: 10-100pM, isopeptidase-T: 10-100nM. Release of AMC fluorescence by DUB enzymes can be monitored using 380 nm excitation and 460 nm emission wavelengths.
Uses[edit]
- Substrate for deubiquitinylating enzyme activity assays.
- Identification/confirmation of enzyme deubiquitinylation activity.
- Investigation of deconjugating enzyme substrate specificity in comparison with alternative UBL-AMC substrates (e.g. NEDD8-AMC)
References[edit]
- ^ BioMol Online Catalog
- ^ Dang, L.C., Melandri, F.D. and Stein, R.L. Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry, 37, 1868-1879 (1998)
- ^ Mason, D.E., Ek, J., Peters, E.C. and Harris J.L. Substrate profiling of deubiquitin hydrolases with a positional scanning library and mass spectrometry. Biochemistry, 43, 6535-44 (2004)