Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Aug;19(6):489-98.
doi: 10.1023/a:1026501515450.

Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate

K E Wee  1 , Z Lai, K R Auger, J Ma, K Y Horiuchi, R L Dowling, C S Dougherty, J I Corman, R Wynn, R A Copeland
Affiliations

Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate

K E Wee et al. J Protein Chem. 2000 Aug.

Abstract

We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl).

PubMed Disclaimer

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Curr Opin Genet Dev. 2000 Feb;10(1):54-64 - PubMed
    1. Cell. 1993 Nov 5;75(3):495-505 - PubMed
    1. J Mol Biol. 1987 Apr 5;194(3):531-44 - PubMed
    1. J Biol Chem. 1982 Mar 10;257(5):2537-42 - PubMed
    1. Anal Biochem. 1986 Apr;154(1):345-52 - PubMed

LinkOut - more resources