Tanaka Hiroyuki, Yamamoto Atsushi, Ishida Tetsuo, Horiike Kihachiro
Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-2192, Japan.
Anal Biochem. 2007 Mar 1;362(1):83-8. doi: 10.1016/j.ab.2006.12.025. Epub 2006 Dec 20.
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.
N-甲基-D-天冬氨酸受体(NMDARs)在脊椎动物中枢神经系统的兴奋性突触传递中起关键作用。NMDARs在各个脑区的通道活动需要D-丝氨酸。在哺乳动物大脑中,D-丝氨酸由丝氨酸消旋酶从L-丝氨酸生成,并由D-氨基酸氧化酶(DAO)降解为3-羟基丙酮酸。在禽类器官如肾脏中,除了DAO外,D-丝氨酸还可被D-丝氨酸脱水酶(DSD)降解为丙酮酸。为了研究这两种酶在禽类大脑中的作用,我们开发了一种同时测量DAO和DSD活性的方法。首先,将D-丝氨酸产生的酮酸用3-甲基-2-苯并噻唑啉酮腙衍生化为稳定的嗪。其次,以2-氧代戊二酸为内标,通过反相高效液相色谱法对嗪衍生物进行定量。该方法能够同时检测低至100 pmol/分钟/毫克蛋白质的DAO和DSD活性。鸡脑仅表现出DSD活性(0.4±0.2 nmol/分钟/毫克蛋白质),而大鼠脑仅表现出DAO活性(0.7±0.1 nmol/分钟/毫克蛋白质)。这一结果有力地表明,DSD在禽类大脑中发挥的作用与DAO在哺乳动物大脑中发挥的作用相同。本方法稍作修改后可应用于其他产生酮酸的酶。