Fluorimetric assay for D-amino acid oxidase activity in rat brain homogenate by using D-kynurenine as a substrate

Abstract

An easy fluorimetric assay for measuring D-amino acid oxidase (DAAO) activity by using one of the D-amino acids--D-kynurenine (D-KYN)--as a substrate was applied to assess DAAO activity in the cerebrum, cerebellum, and brainstem of Sprague-Dawley (SD) male rats. In this assay, DAAO produces kynurenic acid (KYNA) from D-KYN, and the fluorescence originating from KYNA can then be used to evaluate DAAO activity. Here, pellet fractions obtained by centrifugation of brain homogenates were allowed to react enzymatically with D-KYN. The addition of specific DAAO inhibitors, such as 3-methylpyrazole-5-carboxylic acid and 4H-thieno [3, 2-b] pyrrole-5-carboxylic acid (Compound 8), significantly attenuated the fluorescence intensity of KYNA, suggesting that DAAO present in the rat brain homogenates was responsible for the production of KYNA. In contrast, an inhibitor of aminotransferase (AT), aminooxyacetic acid, did not decrease KYNA production from D-KYN, meaning that AT could not metabolize D-KYN to KYNA under the present conditions. Moreover, the DAAO activity measured by the proposed assay correlated well with DAAO mRNA expression (r = 0.9982) determined by real-time polymerase chain reaction. Taken together, these findings show that the proposed fluorimetric assay can be used to evaluate DAAO activity in rat brain.