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

d -氨基酸氧化酶存在于高等动物肾近端小管中。它催化非天然d -氨基酸的氧化脱胺作用。这种酶的生理作用尚不清楚，因为它的底物d -氨基酸被认为在高等动物中很少见。本文对该酶的基因结构和生理功能进行了研究。15株小鼠均能扩增到d -氨基酸氧化酶cDNA。15株中有4株的cDNA hindll识别序列存在单碱基对替换。南方杂交分析表明，15株小鼠携带的d -氨基酸氧化酶基因在整体结构上相似，但其内部结构在小鼠品系之间存在差异。采用原位杂交法检测d -氨基酸氧化酶基因在小鼠肾脏中的表达。该酶的mRNA仅在近端小管上皮细胞中检测到。这一结果表明，这种酶是在近端小管的细胞内合成、保留和起作用的。研究发现，缺乏d -氨基酸氧化酶的突变小鼠会将大量d -丙氨酸和d -丝氨酸排泄到尿液中。研究了这些d -氨基酸的来源。用无菌和非生物突变小鼠进行的实验表明，尿d -丙氨酸来自肠道细菌。抗生素没有减少尿d -丝氨酸，表明它不是细菌来源。营养实验表明，d -丝氨酸的一部分来自老鼠的饮食，但其余部分是内源性的。由于在正常小鼠中未见d -氨基酸性尿症，因此本研究使用d -氨基酸氧化酶缺陷小鼠表明该酶的生理作用之一是d -氨基酸的代谢。

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.