Hassiepen, Ulrich, Eidhoff, Ulf, Meder, Gabriele, Bulber, Jean-Francois, Hein, Andreas, Bodendorf, Ursula, Lorthiois, Edwige and Martoglio, Bruno (2007) A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate. Analytical Biochemistry, 371 (2). pp. 201-207. ISSN 0003-2697

Abstract

The dynamic modification of proteins with ubiquitin is a key regulation paradigm in eukaryotic cells that controls stability, localization, and function of the vast majority of intracellular proteins. Here we describe a robust fluorescence intensity assay for monitoring the enzymatic activity of deubiquitinating proteases, which reverse ubiquitin modifications and comprise over 100 members in humans. The assay was developed for the catalytic domain of human ubiquitin-specific protease 2 (USP2) and human ubiquitin carboxyterminal hydrolase L3 (UCH-L3), and makes use of the novel substrate ubiquitin-rhodamine110-glycine. The latter combines the advantages of a high dynamic range and beneficial optical properties. Its enzymatic behavior is characterized by the kinetic constants K(m)=1.5 microM, k(cat) = 0.53s(-1) and k(cat)/K(m) = 3.5 x 10(5)M(-1) s(-1) for USP2 and K(m) = 34 nM, k(cat)=4.72s(-1), and k(cat)/K(m) = 1.4 x 10(8)M(-1) s(-1) for UCH-L3. This new assay is suitable for inhibitor screening and characterizations, and has been established for the 384-well plate format using protease concentrations of 120 pM for USP2 and 1 pM for UCH-L3 and substrate concentrations of 100 nM for both enzymes. Due to the low protease concentrations and high sensitivity, this assay would allow the determination of inhibitory constants in the subnanomolar range.

Item Type:	Article
Related URLs:	http://www.ncbi.nlm.nih.gov/sites/entrez... doi:10.1016/j.ab.2007.07.034
Additional Information:	author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used
Keywords:	Nedd8; Protease assay; Rhodamine; SUMO; Ubiquitin-specific proteases
Related URLs:	http://www.ncbi.nlm.nih.gov/sites/entrez... doi:10.1016/j.ab.2007.07.034
Date Deposited:	14 Dec 2009 14:00
Last Modified:	31 Jan 2013 01:18
URI:	https://oak.novartis.com/id/eprint/374