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，能催化多种D-氨基酸的氧化脱氨。在哺乳动物中，DAO在肾脏、肝脏和大脑中的浓度最高。关于DAO的基本问题之一是它的生理功能，因为它似乎是一种丰富的酶，而没有天然存在的底物。为了寻找DAO在神经系统中的特异性表达，我们对猪组织进行了RNA印迹杂交分析，结果表明，在肾脏和肝脏中有三种mRNA表达，而在脑中只检测到一种，表明存在脑特异性表达调节。用逆转录酶偶联PCR方法检测两个人脑星形胶质细胞瘤细胞的DAO mRNA，结果均未检测到DAO mRNA，而人小脑组织的双链cDNA中含有高拷贝数的DAO mRNA。培养物在体外2周后汇合，并在第15天进行RNA提取。用1 μ g polyA^+ RNA合成cDNA并进行PCR分析。我们可以检测到预期大小的扩增DNA在培养的皮质星形胶质细胞，以及在小脑和肾脏。尽管DAO已被证实在皮质中不存在，但这些结果是一致的且可重复的，因此表明在皮质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.