Universität zu Köln

Hauser, Christoph, Wodtke, Robert ORCID: 0000-0001-7462-7111, Loeser, Reik and Pietsch, Markus (2017). A fluorescence anisotropy-based assay for determining the activity of tissue transglutaminase. Amino Acids, 49 (3). S. 567 - 584. WIEN: SPRINGER WIEN. ISSN 1438-2199

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Abstract

Tissue transglutaminase (TGase 2) is the most abundantly expressed enzyme of the transglutaminase family and involved in a large variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor growth, progression and metastasis. As a result, TGase 2 represents an attractive target for drug discovery and development, which requires assays that allow for the characterization of modulating agents and are appropriate for high-throughput screening. Herein, we report a fluorescence anisotropy-based approach for the determination of TGase 2's transamidase activity, following the time-dependent increase in fluorescence anisotropy due to the enzyme-catalyzed incorporation of fluoresceinaEuro and rhodamine BaEuroconjugated cadaverines 1-3 (acyl acceptor substrates) into N,N-dimethylated casein (acyl donor substrate). These cadaverine derivatives 1-3 were obtained by solidaEurophase synthesis. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions, such as sarcosyl and isonipecotyl, were introduced between the cadaverine and xanthenyl entities in compounds 2 and 3, respectively, with acyl acceptor 3 showing the most optimal substrate properties of the compounds investigated. The assay was validated for the search of both irreversible and reversible TGase 2 inhibitors using the inactivators iodoacetamide and a recently published laEurolysine-derived acrylamide and the allosteric binder GTP, respectively. In addition, the fluorescence anisotropy-based method was proven to be suitable for high-throughput screening (Z' factor of 0.86) and represents a non-radioactive and highly sensitive assay for determining the active TGase 2 concentration.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Hauser, Christoph UNSPECIFIED UNSPECIFIED UNSPECIFIED Wodtke, Robert UNSPECIFIED orcid.org/0000-0001-7462-7111 UNSPECIFIED Loeser, Reik UNSPECIFIED UNSPECIFIED UNSPECIFIED Pietsch, Markus UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-237598
DOI:	10.1007/s00726-016-2192-5
Journal or Publication Title:	Amino Acids
Volume:	49
Number:	3
Page Range:	S. 567 - 584
Date:	2017
Publisher:	SPRINGER WIEN
Place of Publication:	WIEN
ISSN:	1438-2199
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language PIG LIVER TRANSGLUTAMINASE; PROTEIN-PROTEIN INTERACTIONS; KINETIC-ANALYSIS; CELL-DEATH; CINNAMOYL INHIBITORS; SELECTIVE INHIBITORS; HUNTINGTONS-DISEASE; COLORIMETRIC ASSAY; NUCLEOTIDE-BINDING; SUBSTRATE PEPTIDE Multiple languages Biochemistry & Molecular Biology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/23759