Katane Masumi, Nakayama Kazuki, Kawata Tomonori, Yokoyama Yuki, Matsui Yuto, Kaneko Yuusuke, Matsuda Satsuki, Saitoh Yasuaki, Miyamoto Tetsuya, Sekine Masae, Homma Hiroshi
Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
J Pharm Biomed Anal. 2015 Dec 10;116:109-15. doi: 10.1016/j.jpba.2014.12.037. Epub 2014 Dec 30.
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.
D-天冬氨酸（D-Asp）是一种在哺乳动物体内发现的游离D-氨基酸，在中枢神经、神经内分泌和内分泌系统中发挥着关键作用。在哺乳动物组织中，D-天冬氨酸氧化酶（DDO）是一种立体特异性作用于D-天冬氨酸的降解酶。天冬氨酸消旋酶是一种能从L-天冬氨酸产生D-天冬氨酸的合成酶，已在几种低等生物中被鉴定出来；然而，哺乳动物体内D-天冬氨酸的生物合成途径仍有待充分阐明。本研究的目的是建立一种简单、准确且灵敏的酶法来测定天冬氨酸消旋酶活性。以重组嗜热链球菌天冬氨酸消旋酶作为模型酶，对两种测定天冬氨酸消旋酶活性的酶法进行了优化。在这些方法中，重组人DDO用于将天冬氨酸消旋酶反应由L-天冬氨酸生成的D-天冬氨酸降解为2-氧代酸，然后使用比色法测定其含量。在一种被称为偶联法的方法中，DDO被同时包含在天冬氨酸消旋酶反应混合物中，在孵育过程中，天冬氨酸消旋酶反应很容易通过DDO与D-天冬氨酸降解反应偶联。在另一种被称为分离法的方法中，天冬氨酸消旋酶反应混合物的一份等分试样在反应后与DDO混合，以测定天冬氨酸消旋酶产生的D-天冬氨酸含量。在优化条件下，对这两种方法的准确性和灵敏度进行了相互比较，并与传统的高效液相色谱法（HPLC）进行了比较。此处给出的结果表明，偶联法比其他两种方法更准确、灵敏，可用于测定天冬氨酸消旋酶活性。偶联法可能有助于推进我们目前对哺乳动物体内D-天冬氨酸生物合成途径的理解。
