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Elucidation of gene structure and physiological function of D-amino-acid oxidase present in the proximal tubules of the kidney

阐明肾近曲小管中 D-氨基酸氧化酶的基因结构和生理功能

基本信息

批准号：
05670959
负责人：
KONNO Ryuichi
金额：
$ 1.41万
依托单位：
Dokkyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

D-Amino-acid oxidase is present in the proximal tubules of the kidneys of higher animals. It catalyzes oxidative deamination of unnatural D-amino acids. The physiological roles of this enzyme have been unknown because its substrate D-amino acids are considered to be rare in higher animals. The gene structure and physiological functions of this enzyme have been examined in this study.D-Amino-acid oxidase cDNA was able to be amplified by PCR in all the mice of 15 strains examined. There was a single-base pair substitution in the Hindlll recognition sequence in the cDNA in four out of the 15 strains.The Southern hybridization analyzes have shown that the 15 strains of mice carry the D-amino-acid oxidase gene similar in the whole structure but also shown that there are variations in its inside structure among the mouse strains.The expression of the D-amino-acid oxidase gene in the mouse kidney was examined by in situ hybridization. mRNA for this enzyme was only detected in the epithelial cells of the proximal tubules. This result indicates that this enzyme is synthesized, remains, and functions within the cells of the proximal tubules.The mutant mice lacking D-amino-acid oxidase were found to excrete a large amounts of D-alanine and D-serine into their urine. The sources of these D-amino acids were examined. Experiments using the germ-free and gnotobiotic mutant mice have demonstrated that the urinary D-alanine comes from intestinal bacteria. Antibiotic administration did not reduce the urinary D-serine, indicating it was not of bacterial origin. Nutritional experiments have shown that some part of the D-serine comes from the mouse diet but the rest of it has an endogenous origin. Since D-aminoaciduria is not seen in normal mice, the present study using D-amino-acid oxidase-deficient mice indicates one of the physiological roles of this enzyme is the metabolism of D-amino acids.

D-氨基酸氧化酶存在于高等动物肾脏的近端小管中。它催化非天然D-氨基酸的氧化脱氨。这种酶的生理作用一直不清楚，因为它的底物D-氨基酸在高等动物中被认为是罕见的。本研究对该酶的基因结构和生理功能进行了研究，15个品系的小鼠均能通过PCR扩增出D-氨基酸氧化酶的基因片段。在15个品系中，有4个品系的Hindll识别序列存在单碱基替换。Southern杂交分析表明，这15个品系小鼠的D-氨基酸氧化酶基因在整体结构上相似，但其内部结构在不同品系之间存在差异。该酶的mRNA仅在近端小管上皮细胞中检测到。这一结果表明，该酶在近端小管细胞内合成、保留和发挥功能。突变的D-氨基酸氧化酶缺失的小鼠被发现将大量的D-丙氨酸和D-丝氨酸排泄到它们的尿液中。对这些D-氨基酸的来源进行了探讨。使用无菌和诺生突变小鼠的实验证明，尿液中的D-丙氨酸来自肠道细菌。服用抗生素并没有减少尿液中的D-丝氨酸，这表明它不是细菌造成的。营养实验表明，D-丝氨酸的一部分来自老鼠的饮食，但其余的来自内源性来源。由于D-氨基酸尿症在正常小鼠中不存在，目前在D-氨基酸氧化酶缺陷小鼠身上的研究表明，该酶的生理作用之一是D-氨基酸的代谢。