Joint Study on the Function of Brain D-Amino Acid Oxidase

D-氨基酸氧化酶(DAO)是一种以FAD为辅基的黄素酶，催化多种D-氨基酸的氧化脱氨反应。在哺乳动物中，DAO在肾脏、肝脏和脑中的浓度最高。关于DAO的一个基本问题是它的生理功能，因为它看起来是一种丰富的酶，没有自然产生的底物。最近有报道指出，D-丝氨酸存在于脑中，并且DAO在脑中的分布与D-丝氨酸的分布呈负相关。为了寻找DAO在神经系统中的特异性表达，对猪组织进行了RNA印迹杂交分析，发现有三种基因在肾脏和肝脏表达，而在脑中只有一种基因表达，表明存在脑特异性表达调控。两种人脑星形细胞瘤细胞的逆转录酶偶联聚合酶链式反应(RT-PCR)检测不到DAO的mRNA.但人小脑组织的双链cDNA中含有高拷贝数的DAO mRNA.为了进一步研究大脑DAO的表达，从出生后1-2天的幼鼠大脑皮层制备了原代培养的星形胶质细胞.体外培养2周后融合，第15天进行RNA提取，1杯Polya^+RNA合成cDNA，进行聚合酶链式反应分析。我们可以在培养的大脑皮层星形胶质细胞以及小脑和肾脏中检测到预期大小的扩增DNA。虽然已有报道称大脑皮层中没有DAO，但这些结果是一致的和可重复性的，因此表明大脑皮层星形胶质细胞中存在DAO信息。

D-amino acid oxidase (DAO) is a flavoenzyme with FAD as its prosthetic group that catalyzed the oxidative deamination of a wide range of D-amino acids. In mammals, DAO is found in highest concentrations in kidney, liver and brain. One of the fundamental questions regarding DAO is that of its physiological function, since it woud appear to be an abundant enzyme without a naturally occuringd substrate. Of particular interest are recent reports that D-serine is present in brain and the distribution of DAO in the brain is inversely correlated to that of D-serine.In search of nervous system specific expression of DAO,RNA blot hybridization analysis of porcine tissues was carried out to show that three mRNA species were expressed in kidney and liver, but only one was detected in brain, indicationg the presence of brain specific regulation of the expression. The reverse transcriptase-coupled PCR analyzes of two human astrocytoma cells derived from cerebral cortex could not detect DAO mRNA.However, double-stranded cDNA of human cerebellar tissue contained high copy number of DAO mRNA.In ordr to investigate further the expression of cerebral DAO,primary cortical astrocytic cultures were prepared from cerebral cortex isolated from post natal rat pups at day 1-2. The cultures were confluent after 2 weeks in vitro and RNA extraction was performed at day 15. cDNA was synthesized from 1 mug of polyA^+ RNA and PCR analysis was performed. We could detect the amplified DNA of the expected size in cultured cortical astrocytes as well as in cerebellum and kidney. Although DAO has been reporated to be absent in cortex, these results were consistent and reproducible thus indicating the presence of the DAO message in cortical astrocytes.