Methods for the Detection of D-Amino-Acid Oxidase-2

D-Amino-acid oxidase activity was detected in the kidneys of human, monkey, rat, mouse, chicken, frog, carp, dace, crucian carp, cat fish, rainbow trout, and electric ray (10, 11). D-Amino-acid oxidase protein was detected by western blotting in the kidney and brain of the mouse, and in the kidney of the rat (12-14). RT-PCR amplified a D-amino-acid oxidase cDNA fragment from RNA extracted from the kidney and brain of the mouse, and from the kidney, liver, and cerebellum of the rat (13, 14). Northern hybridization showed the presence of mRNA for D-amino-acid oxidase in the kidney and cerebellum of the mouse, and in the kidney, liver, and hindbrain of the rat (13, 14).

All the vertebrates examined so far have D-amino-acid oxidase in their livers (1, 2, 15, 16). However, the mouse liver did not show positive results in the D-amino-acid oxidase enzyme activity assay, western blotting, RT-PCR, and northern hybridization. Therefore, we concluded that the mouse does not have this enzyme in its liver (13). The mouse is a very unique animal in this respect.

Northern hybridization, RT-PCR, and western blotting gave positive results for the presence of D-amino-acid oxidase in the ddY/DAO- mice (12, 17). However, the kidney and brain homogenates of these mice did not show D-amino-acid oxidase activity (18, 19). Therefore, we concluded that they produced the D-amino-acid oxidase protein without enzyme activity. This lack of activity was due to a single-base substitution in the coding region of the cDNA for this enzyme (17).