Development of a microplate fluorescence assay for kynurenine aminotransferase

Inhibition of kynurenine aminotransferases (KATs) is a strategy to therapeutically reduce levels of kynurenic acid (KYNA), an endogenous antagonist of glutamatergic N-methyl- d-aspartate (NMDA) and cholinergic α 7 nicotinic receptors. Several methods of measuring KAT activity in vitro have been deve...

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Bibliographic Details
Published in:Analytical biochemistry 2011-02, Vol.409 (2), p.183-188
Main Authors: Wong, Jacky, Ray, William J., Kornilova, Anna Y.
Format: Article
Language:English
Subjects:
Cells, Cultured
Enzymatic assay
Enzyme Assays - methods
Fluorescence
HTS
Humans
Hydrogen-Ion Concentration
Inhibitors
Kynurenic Acid - metabolism
Kynurenine aminotransferase
Spectrometry, Fluorescence
Transaminases - analysis
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Summary:Inhibition of kynurenine aminotransferases (KATs) is a strategy to therapeutically reduce levels of kynurenic acid (KYNA), an endogenous antagonist of glutamatergic N-methyl- d-aspartate (NMDA) and cholinergic α 7 nicotinic receptors. Several methods of measuring KAT activity in vitro have been developed, but none is well-suited to high throughput and automation. In this article, we describe a modification of existing high-performance liquid chromatography (HPLC)-based methods that enables the development of a 96-well microplate assay in both enzyme- and cell-based formats using human KAT I as an example. KYNA enzymatically produced from l-kynurenine is measured directly in a reaction mixture fluorimetrically.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2010.10.037