Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate

Thangaraju, Kiruphagaran and Biri, Beáta and Schlosser, Gitta (Vácziné) and Kiss, Bence and Nyitray, László and Fésüs, László and Király, Róbert (2016) Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate. Analytical Biochemistry, 505. pp. 36-42. ISSN 0003-2697

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Abstract

Transglutaminase 2 (TG2) is a ubiquitously expressed multifunctional protein with Ca2+-dependent transamidase activity forming protease resistant Nε-(γ-glutamyl)lysine crosslinks between proteins. It can also function as an isopeptidase cleaving the previously formed crosslinks. The biological significance of this activity has not been revealed yet mainly because of the lack of protein based method for its characterization. Here we report development of a novel kinetic method for measuring isopeptidase activity of human TG2 by monitoring decrease in the fluorescence polarisation of a protein substrate previously formed by crosslinking fluorescently labelled glutamine donor FLpepT26 to S100A4 at a specific lysine residue. The developed method could be applied to test mutant enzymes and compounds which influence isopeptidase activity of TG2.

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