Simultaneous measurement of d-serine dehydratase and d-amino acid oxidase activities by the detection of 2-oxo-acid formation with reverse-phase high-performance liquid chromatography

Abstract

N-methyl- d-aspartate receptors (NMDARs) play critical roles in excitatory synaptic transmission in the vertebrate central nervous system. NMDARs need d-serine for their channel activities in various brain regions. In mammalian brains, d-serine is produced from l-serine by serine racemase and degraded by d-amino acid oxidase (DAO) to 3-hydroxypyruvate. In avian organs, such as the kidney, in addition to DAO, d-serine is also degraded to pyruvate by d-serine dehydratase (DSD). To examine the roles of these two enzymes in avian brains, we developed a method to simultaneously measure DAO and DSD activities. First, the keto acids produced from d-serine were derivatized with 3-methyl-2-benzothiazolinone hydrazone to stable azines. Second, the azine derivatives were quantified by means of reverse-phase high-performance liquid chromatography using 2-oxoglutarate as an internal standard. This method allowed the simultaneous detection of DAO and DSD activities as low as 100 pmol/min/mg protein. Chicken brain showed only DSD activities (0.4 ± 0.2 nmol/min/mg protein) whereas rat brain exhibited only DAO activities (0.7 ± 0.1 nmol/min/mg protein). This result strongly suggests that DSD plays the same role in avian brains, as DAO plays in mammalian brains. The present method is applicable to other keto acids producing enzymes with minor modifications.