UCHL3, human recombinant

Catalog number: SBB-DE0023, 50 μg

UCHL3 is a deubiquitinating enzyme (DUB) that controls levels of cellular ubiquitin through processing of ubiquitin precursors and ubiquitinated proteins. It is a thiol protease that recognizes and hydrolyzes a peptide bond at the C-terminal glycine of either ubiquitin or NEDD8. It digests precursors and ubiquitinated proteins to generate monomeric ubiquitin. This UCHL3 is recombinatly expressed in E.coli.[1][2][3][4][5]

In stock
SKU
0023
$149.00

Description

UCHL3 (Ubiquitin C-terminal Hydrolase L3) is a deubiquitinating enzyme (DUB) that controls levels of cellular ubiquitin through processing of ubiquitin precursors and ubiquitinated proteins. It is a thiol protease that recognizes and hydrolyzes a peptide bond at the C-terminal glycine of either ubiquitin or NEDD8. It plays a role in regulating apical membrane recycling, and indirectly increases the phosphorylation of IGFIR, AKT and FOXO1 and promotes insulin-signaling and insulin-induced adipogenesis. It is required for stress-response in retinal, skeletal muscle and germ cell maintenance. UCHL3 is also known to hydrolyze UBB(+1), a mutated form of ubiquitin which is not effectively degraded by the proteasome and is associated with neurogenerative disorders. It digests precursors and ubiquitinated proteins to generate monomeric ubiquitin. This UCHL3 is recombinatly expressed in E.coli.

For Research Use Only, Not For Use In Humans.

Specifications

Product Overview
Quantity: 50 μg
Molecular Weight: 26 kDa
Purity: >95% by SDS-PAGE
Substrate Properties: Working concentrations of this enzyme range from 0.5 to 5nM.
Storage Buffer: 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM TCEP
Storage Store at −80°C after product arrival. Avoid multiple freeze / thaws. It is recommended to make multiple aliquots after the first thaw.

Figures & Data

UCHL3, human recombinant

Figure 1. UCHL3, SDS-PAGE From left to right, increasing amounts of UCHL3 loaded onto a 4-12% SDS-PAGE gel, stained with Coomassie blue.

UCHL3 Michaelis–Menten Kinetics

Figure 2. Michaelis–Menten Kinetics. Ubiquitin Rhodamine 110 serially diluted from 1.6 to 0.05 uM was digested with 30pM UCHL3 over time. The assay was carried out in a reaction buffer of 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM TCEP, 0.1 mg/ml BSA, at 25C. Initial velocities at each substrate concentration were plotted and fit to the Michaelis–Menten equation. Kinetic parameters were calculated at: Km= 0.69 μM, Vmax = 1.29 nMs-1, kcat= 43s-1, kcat/Km= 6.16 x 107 M-1s-1.

Citations & References

1) Wilkinson K.D., Lee K., Deshpande S., Duerksen-Hughes P., Boss J.M., Pohl J. "The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase." Science 246:670-673(1989)

2) Wada H., Kito K., Caskey L.S., Yeh E.T.H., Kamitani T. "Cleavage of the C-terminus of NEDD8 by UCH-L3." Biochem. Biophys. Res. Commun. 251:688-692(1998)

3) Setsuie R., Sakurai M., Sakaguchi Y., Wada K. "Ubiquitin dimers control the hydrolase activity of UCH-L3." Neurochem. Int. 54:314-321(2009)

4) Dennissen F.J., Kholod N., Hermes D.J., Kemmerling N., Steinbusch H.W., Dantuma N.P., van Leeuwen F.W. "Mutant ubiquitin (UBB(+1)) associated with neurodegenerative disorders is hydrolyzed by ubiquitin C-terminal hydrolase L3 (UCH-L3)." FEBS Lett. 585:2568-2574(2011)

5) Iphofer A., Kummer A., Nimtz M., Ritter A., Arnold T., Frank R., van den Heuvel J., Kessler B.M., Jansch L., Franke R. "Profiling ubiquitin linkage specificities of deubiquitinating enzymes with branched ubiquitin isopeptide probes." ChemBioChem 13:1416-1420(2012)