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

d -氨基酸氧化酶（DAO）是一种以FAD为假基的黄酶，可催化多种d -氨基酸的氧化脱胺反应。在哺乳动物中，DAO在肾脏、肝脏和大脑中含量最高。关于DAO的一个基本问题是它的生理功能，因为它似乎是一种丰富的酶，没有自然存在的底物。特别令人感兴趣的是最近的报道，d -丝氨酸存在于大脑中，DAO在大脑中的分布与d -丝氨酸的分布呈负相关。为了寻找DAO的神经系统特异性表达，我们对猪组织进行了RNA印迹杂交分析，结果显示，在肾脏和肝脏中有3种mRNA表达，而在大脑中只检测到1种mRNA，表明存在大脑特异性表达调控。用逆转录酶偶联PCR对2个人类大脑皮层星形细胞瘤细胞进行分析，无法检测到DAO mRNA。然而，人小脑组织的双链cDNA含有高拷贝数的DAO mRNA。为了进一步研究大脑DAO的表达，我们在1-2天从出生后的大鼠幼崽的大脑皮层分离制备了初级皮层星形细胞培养物。体外培养2周后融合，第15天进行RNA提取。从1杯的polyA^+ RNA中合成cDNA并进行PCR分析。我们在培养的皮质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。虽然有报道称DAO在皮层中不存在，但这些结果是一致的和可重复的，因此表明DAO信息存在于皮层星形胶质细胞中。

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.