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 α₇ 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.
Neurology Department, Merck, West Point, PA 19486, USA.
Jacky
J
Wong
William J
WJ
Ray
Anna Y
AY
Kornilova
D002478:Cells, Cultured; D057075:Enzyme Assays / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007736:Kynurenic Acid / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D000637:Transaminases / Q000032:analysis*
D000637:Transaminases; C021481:kynurenine-oxoglutarate transaminase; D007736:Kynurenic Acid