title: A sensitive assay for measuring aspartate-specific amino acid racemase activity.
abstract: D-Aspartate (D-Asp), a free D-amino acid found in mammals, plays crucial roles in the central nervous, neuroendocrine, and endocrine systems. In mammalian tissues, D-Asp oxidase (DDO) is a degradative enzyme that stereospecifically acts on D-Asp. Asp racemase, a synthetic enzyme that produces D-Asp from L-Asp, has been identified in several lower organisms; however, the biosynthetic pathway of D-Asp in mammals remains to be fully clarified. The aim of this study was to establish a simple, accurate, and sensitive enzymatic method for the determination of Asp racemase activity. Using recombinant Streptococcus thermophilus Asp racemase as a model enzyme, two enzymatic methods for the determination of Asp racemase activity were optimized. In these methods, recombinant human DDO was used to degrade D-Asp formed from L-Asp by the Asp racemase reaction to 2-oxo acid, the amounts of which were then determined using a colorimetric assay. In one method, designated the coupling method, DDO was concomitantly included in the Asp racemase reaction mixture, and the Asp racemase reaction was readily coupled to the D-Asp degradative reaction by DDO during the incubation. In the other method, designated the separating method, an aliquot of the Asp racemase reaction mixture was mixed with DDO after the reaction to determine the amounts of D-Asp produced by Asp racemase. Under optimized conditions, the accuracy and sensitivity of these two methods were examined and compared, both to one another and conventional high-performance liquid chromatography (HPLC). The results presented here suggest that the coupling method is more accurate and sensitive than the other two methods and can be used for the determination of Asp racemase activity. The coupling method may help to advance our current understanding of the biosynthetic pathway of D-Asp in mammals.
authors.affiliation: Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Masumi
authors.initials: M
authors.lastname: Katane
authors.affiliation: Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Nakayama
authors.affiliation: Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Kawata
authors.affiliation: Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yokoyama
authors.affiliation: Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Yuto
authors.initials: Y
authors.lastname: Matsui
authors.affiliation: Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Yuusuke
authors.initials: Y
authors.lastname: Kaneko
authors.affiliation: Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Satsuki
authors.initials: S
authors.lastname: Matsuda
authors.affiliation: Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Saitoh
authors.affiliation: Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Miyamoto
authors.affiliation: Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Masae
authors.initials: M
authors.lastname: Sekine
authors.affiliation: Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address: hommah@pharm.kitasato-u.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Homma
mesh_terms: D000591:Amino Acid Isomerases / Q000032:analysis* / Q000378:metabolism*; D001224:Aspartic Acid / Q000032:analysis* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D048249:Streptococcus thermophilus / Q000201:enzymology
chemical_list: D001224:Aspartic Acid; D000591:Amino Acid Isomerases; C019749:aspartate racemase
keywords: Amino acid racemase; Aspartate racemase; Enzymatic method; d-Aspartate; d-Aspartate oxidase