Biosynthesis of D-aspartate in mammalian cells

In this communication, we demonstrate that D-aspartate (D-Asp) is synthesized in pheochromocytoma cells (PC12). To our knowledge this is the first report of biosynthesis of D-Asp in mammalian cells. Synthesis of D-Asp was demonstrated by its time-dependent accumulation in the cell culture, and by the fact that this accumulation was proportional to the number of inoculated cells. D-Asp in PC12 cells was identified by (i) co-elution with authentic D-Asp on two different HPLC columns, an octadesyl silica column and a Pirkle-type chiral column, (ii) reversed elution order of D-Asp and L-Asp on another Pirkle-type chiral column with an opposite configuration, and (iii) sensitivity to D-Asp oxidase. In the cells the amount of D-Asp was approx. 12-14% of total Asp and no other investigated D-amino acid was detected. The amount of D-Asp did not increase during the culture of mouse 3T3 fibroblasts and human neuroblastoma NB-1 cells. Immunocytochemical staining with anti-D-Asp antiserum demonstrated that D-Asp synthesized is present in the cytoplasm of the cells.