Development of a microplate fluorescence assay for kynurenine aminotransferase.

Abstract 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.