A highly sensitive enzymatic assay for d- and total serine detection using D-serine dehydratase from Saccharomyces cerevisiae

摘要

0-Serine acts as a co-agonist of the N-methyl-D-aspartate (NMDA) receptor, an excitatory glutamate receptor in the mammalian brain that is thought to be involved in higher brain functions such as memory and study. A relationship between the concentration of D-serine in cerebrospinal fluid and neurological disorders such as schizophrenia and amyotrophic lateral sclerosis has been hypothesized. A simple and accurate D-serine assay system would be useful in the study and diagnosis of these diseases. Previously, we developed an enzymatic assay of D-serine using D-serine dehydratase from Saccharomyces cerevisiae, lactate dehydrogenase and NADH. Although this method can detect 10μM D-serine, a 10-fold increase in sensitivity is required for the assay to be useful for the study and diagnosis of neurological disorders. In this study, we increased the sensitivity of this assay to detect submicromolar concentrations of d-serine. In the new assay, D-serine dehydratase converts D-serine to pyruvate, which is in turn oxidized by pyruvate oxidase. Then, in the presence of horseradish peroxidase, hydrogen peroxide formed during the oxidation converts 10-acetyl-3,7-dihydroxy-phenoxazine (Amplex~R Red) to resorufin, which exhibits a strong fluorescence. We show that this improved assay can be used to determine the concentration of D-serine in calf serum.