Figure 2.

Serine racemase catalyses the formation of d-serine in vivo. (A) Analysis of d-serine synthesis in transfected cells. HEK293 cells were transfected either with full-length mouse serine racemase (○) or serine racemase mutant K56G (●). (B) Analysis of d-serine synthesis in culture media from transfected cells. (C) Synthesis of d-serine in cell homogenates. Mock-transfected cells or K56G mutant exhibited no activity, whereas addition of 0.5 mM amino-oxyacetic acid (AOAA) inhibited most of the activity in serine racemase-transfected cell extracts. (D–G) HEK293 cells were transfected with serine racemase and further analyzed for d-serine content by immunocytochemistry. (D) Mock-transfected cells incubated in DMEM media supplemented with 10 mM l-serine. (E) Serine racemase-transfected cells incubated in media supplemented with 10 mM l-serine. (F) Serine racemase-transfected cells incubated in media supplemented with 10 mM l-serine and stained with antibody preabsorbed with 0.5 mM d-serine-glutaraldehyde conjugate. (G) Mock-transfected cells first incubated for 12 hr in media containing 5 mM d-serine. Intense immunostaining represents accumulation of d-serine by the cells, which was not significantly metabolized during the course of the experiment. (H and I) Synthesis of d-serine by mixed neuronal-glia cell culture. A significant increase in d-serine synthesis was observed by supplementing the media with 5 mM l-serine. d-serine produced in cells (H) and released to culture media (I) was analyzed 48 hr after addition of l-serine. The results are representative of four independent experiments with different culture preparations (A–G) and presented as the mean ± SEM of three independent experiments (H and I).