Biosynthesis of the Neuromodulator D-Serine

Proc. Natl. Acad. Sci. USA Vol. 96, pp. 721–725, January 1999 Neurobiology

Purification of serine racemase: Biosynthesis of the neuromodulator D-serine

HERMAN WOLOSKER,KEVIN N. SHETH,MASAAKI TAKAHASHI,JEAN-PIERRE MOTHET,ROSCOE O. BRADY,JR., CHRISTOPHER D. FERRIS, AND SOLOMON H. SNYDER*

The Johns Hopkins University, School of Medicine, Departments of Neuroscience, Pharmacology and Molecular Sciences, and Psychiatry, 725 North Wolfe Street, Baltimore, MD 21205

Contributed by Solomon H. Snyder, December 2, 1998

ABSTRACT High levels of D-serine occur in mammalian than any direct enzymatic racemization. In the present study brain, where it appears to be an endogenous ligand of the we describe an enzyme activity that directly racemizes L-serine glycine site of N-methyl-D-aspartate receptors. In glial cul- to D-serine and that is localized to the brain. We report tures of rat cerebral cortex, D-serine is enriched in type II purification to homogeneity of this enzyme and its require- astrocytes and is released upon stimulation with agonists of ment for pyridoxal phosphate. non-N-methyl-D-aspartate glutamate receptors. The high levels of D-serine in discrete areas of rat brain imply the existence MATERIALS AND METHODS of a biosynthetic pathway. We have purified from rat brain a soluble enzyme that catalyzes the direct racemization of Materials. Amino acids, aminooxyacetic acid (AOAA), L-serine to D-serine. Purified serine racemase has a molecular catalase, oxidized glutathione, hydroxylamine, leupeptin, lu- ,mass of 37 kDa and requires pyridoxal 5؅-phosphate for its minol (sodium salt), pepstatin, phenylmethylsulfonyl fluoride activity. The enzyme is highly selective toward L-serine, failing pyridoxal 5Ј-phosphate (PLP), o-phthaldialdehyde (OPA), to racemize any other amino acid tested. Properties such as pH L-homocysteic acid, and Tris were obtained from Sigma. optimum, Km values, and the requirement for pyridoxal phos- D-Amino acid oxidase from pig kidney (EC 1.4.3.3), DTT, and phate resemble those of bacterial racemases, suggesting that horseradish peroxidase were obtained from Boehringer Mann- the biosynthetic pathway for D-amino acids is conserved from heim. Ammonium sulfate, KH2PO4, and KH2PO4 were pur- bacteria to mammalian brain. chased from J. T. Baker. Butyl Sepharose 4 fast flow, Q-Sepharose, and Mono Q HR 5͞5 were obtained from ␮ D-Amino acids are prominent in bacteria whereas in animal Pharmacia. Macro-prep ceramic hydroxyapatite type I (20 m) tissues L-amino acids occur exclusively, though there have been was purchased from Bio-Rad. N-tert-butyloxycarbonyl-L- occasional reports of D-amino acids, usually in invertebrates (1, cysteine (L-Boc-cys) was obtained from Nova Biochem. Other 2). Recently, D-serine (3–6) and D-aspartate (7, 8) were reagents were of analytical grade. reported in mammalian tissues, especially in the nervous Assay of Serine Racemase. D-serine formation was moni- system. Using highly selective antibodies, we localized tored by a chemiluminescent assay that specifically detects D-aspartate to neuroendocrine tissues (9), whereas the immu- D-serine. Racemase activity was performed in the presence of 50 mM Tris⅐HCl, pH 8.0͞18 ␮l enzyme extract͞1 mM EDTA͞2 nohistochemical localizations of D-serine closely resemble ͞ ␮ ͞ N-methyl-D-aspartate (NMDA) receptors for the neurotrans- mM DTT 15 M PLP 20 mM L-serine. After 0.5–8 h of mitter glutamate, as the distribution of D-serine measured incubation at 37°C, the reaction was terminated by the addition chemically (10, 11). Glutamate cannot activate the NMDA of trichloroacetic acid (TCA) to a final concentration of 5%. receptor in the absence of glycine, indicating a ‘‘glycine site’’ Blanks used boiled enzyme extract. The precipitated protein for the receptor (12, 13). D-Serine is up to three times more was removed by centrifugation, and the supernatant was potent than glycine at this site (14), suggesting that D-serine is extracted two times with 1 ml of water-saturated diethyl ether D the endogenous ligand for this site. D-Serine is localized to remove TCA. -Serine was determined by incubation of the samples with D-amino acid oxidase, which specifically degrades exclusively to type II astrocytes, a form of glia concentrated in ␣ gray matter in the same areas of the brain as NMDA receptors D-amino acids, generating an -keto acid, NH3, and hydrogen (10). Stimulation of the kainate subtype of glutamate receptors peroxide (16). The generation of hydrogen peroxide was quantitated by the use of peroxidase and luminol, which emits releases D-serine from type II astrocytes, implying that syn- light. A 10-␮l sample aliquot was added to 100 ␮l of medium aptic release of glutamate triggers release of D-serine from the ⅐ astrocytes to activate NMDA receptors physiologically (10). containing 100 mM Tris HCl, pH 8.8, 10 units/ml peroxidase, and 8 ␮M luminol. After a 10- to 20-min delay, required to Although in most parts of the brain the distribution of D-serine decrease the nonspecific luminol luminescence, 10 ␮lofD- resembles NMDA receptors far better than glycine, in some ͞ areas glycine and NMDA receptors are colocalized, suggesting amino acid oxidase (75 units ml) was added and the tubes were mixed gently with a pipette tip. Maximum luminescence was that D-serine is the predominant ligand for the receptor in most brain areas but that glycine serves this purpose in some sites recorded after 10–15 min at room temperature by using a (11). Monolight 2010 luminometer (Analytical Luminescence Lab- oratory). The amount of D-serine in each sample was calcu- Understanding the neurobiology of D-serine requires delin- lated by comparing with standard curves. The measurements eation of its biosynthesis. D-Serine might be formed by direct were reliable in the range of 50–2,000 pmol D-serine per racemization from L-serine. Dunlop and Neidle (15) reported sample. Addition of mM concentrations of L-serine did not the transformation of radiolabeled L-serine to D-serine in intact rats, but this might have involved multiple steps rather alter the values measured for D-serine. Alternatively, amino acid enantiomers were separated by HPLC by using a carbon

The publication costs of this article were defrayed in part by page charge Abbreviations: AOAA, aminooxyacetic acid; NMDA, N-methyl-D- payment. This article must therefore be hereby marked ‘‘advertisement’’ in aspartate; PLP, pyridoxal 5Ј-phosphate; TCA, trichloroacetic acid. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. e-mail: ssnyder@ PNAS is available online at www.pnas.org. jhmi.edu.

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Table 1. Purification of serine racemase Specific activity, Fraction Protein, mg ␮mol L-Ser/mg per h Fold purification Total activity Yield, % Homogenate 4,744 ND — — — (NH4)2SO4 fractionation 624 0.0004 1 0.249 100 Butyl-sepharose 34 0.003 7.5 0.102 41 Q-sepharose 6.6 0.029 73 0.191 77 Mono Q 0.22 0.833 2,082 0.183 73 Hydroxyapatite 0.015 5.0 12,500 0.075 30 Enzyme was purified and fractions were assayed as described. Data represent a typical purification, which was repeated six times with similar results. ND, not detected.

18 reverse-phase column (RP18 Spheri-5, 22 cm ϫ 4.6 mm; then centrifuged at 20,000 ϫ g for 20 min to remove insoluble Perkin–Elmer) with fluorimetric detection after derivatization aggregates. The supernatant was loaded at 3 ml͞min onto a with L-Boc-cys and OPA, as described (17). The results 70-ml butyl-Sepharose column preequilibrated with 20% am- obtained with the chemiluminescent assay were identical to monium sulfate in buffer A. The column was washed with 210 those obtained using HPLC. The presence of trace amounts of ml of 10% ammonium sulfate, and the active fraction was D-serine in the commercial L-serine reagent generates high eluted with 5% ammonium sulfate in buffer A. The eluted blank values. Thus, the stock solution of L-serine (100 mM) was material was concentrated by precipitation with 50% ammo- ϫ pretreated routinely for 3 days with 30 units of D-amino acid nium sulfate and centrifugation at 20,000 g for 20 min. The oxidase and 500 units of catalase to remove any D-serine pellet was resuspended in 6–8 ml buffer A and dialyzed ͞ contaminant. The enzymes were precipitated by the addition overnight against 4 liters of buffer B (10 mM KPi, pH 7.2 50 mM KCl͞1 mM EDTA͞2mMDTT͞15 ␮M PLP). After of 5% TCA. After removal of TCA by extraction with diethyl ϫ ether, L-serine solution was neutralized with NaOH and could dialysis, the suspension was centrifuged at 20,000 g for 20 be used without further purification, being virtually free of any min to remove insoluble aggregates, and the supernatant was loaded at 0.5 ml͞min onto a 3-ml Q-Sepharose column. After D-serine contaminant. The same purification procedure was washing with 10 ml loading buffer, the protein was eluted with applied for L-alanine and L-threonine. 250 mM NaCl in buffer B. The eluted material was concen- Purification of Serine Racemase. Sixty brains from 10- to trated with centriprep 30 (Amicon) and diluted in buffer B 14-day-old Sprague–Dawley rats were homogenized by using a without KCl to decrease the salt concentration to 50 mM. Polytron in 5 vol of ice-cold buffer A (10 mM KPi, pH 7.2͞50 Then, the suspension was loaded at 0.5 ml͞min onto a Mono mM KCl͞1 mM EDTA͞2mMDTT͞15 ␮M PLP͞0.2 mM ͞ ␮ Q column. The column was washed with buffer B containing freshly prepared phenylmethylsulfonyl fluoride 1 g/ml leu- 150 mM KCl, and the protein was eluted with a linear gradient peptin͞1 ␮g/ml pepstatin). All subsequent steps were per- ϫ of KCl in the range of 158–188 mM KCl. The active fractions formed at 4°C. The homogenate was centrifuged at 40,000 were pooled, concentrated with centriplus 30 (Amicon), and g for 20 min, and the supernatant was brought to 45% diluted in buffer B without EDTA and KPi. The procedure was ammonium sulfate saturation under continuous stirring. After repeated once to decrease the EDTA and KPi concentrations ϫ ␮ 40-min precipitation, the solution was centrifuged at 20,000 to 20 M and 0.75 mM, respectively. To this suspension, CaCl2 g for 20 min. The pellet was resuspended in 20% ammonium was added (300 ␮M final concentration) to improve the sulfate in buffer A. The suspension was left on ice for1hand protein binding to hydroxyapatite. The protein was applied at 0.1 ml͞min to a 1-ml hydroxyapatite column and eluted with a linear gradient of 0.75–400 mM KPi in buffer containing 50 mM KCl, 2 mM DTT, and 15 ␮M PLP. The purified protein typically was eluted in the range of 0.75–30 mM KPi. For most applications the purified protein was concentrated further using centriplus 30. Protein concentration was determined with Coomassie blue plus protein assay reagent (Pierce). Spectrophotometric Assays. Enzyme-bound PLP was determined at room temperature by recording the absorbance of the purified protein in the range of 500–280 nm using a Lambda Bio spectrophotometer (Perkin–Elmer).

RESULTS Uo et al. (18) recently described serine racemase activity in silkworms and purified the activity approximately 5-fold. They failed to detect serine racemase activity in crude homogenates, but observed activity in a fraction partially purified by ammonium sulfate fractionation. Similarly, in rat brain we cannot detect the conversion of L-serine to D-serine, monitored by HPLC, in homogenates, but can demonstrate this activity in a FIG. 1. SDS͞PAGE analysis of purified serine racemase. A 12% fraction precipitated with 45% ammonium sulfate (Table 1). polyacrylamide gel was stained with Coomassie blue. Lane 1, molec- With the partially purified enzyme, a simple routine assay ular mass markers: myosin (200 kDa), ␤-galactosidase (116.7 kDa), monitors the conversion by added D-amino acid oxidase of phosphorylase b (97.4 kDa), BSA (66.3 kDa), glutamic dehydrogenase ␣ (55.4 kDa), lactate dehydrogenase (36.5 kDa), carbonic anhydrase (31 D-serine to an keto acid and H2O2.H2O2 then is transformed kDa), and trypsin inhibitor (21.5 kDa). Lane 2, Mono Q column eluate into a luminescent compound in the presence of peroxidase containing 1 ␮g protein. Lane 3, hydroxyapatite column eluate and luminol. containing 0.5 ␮g purified protein. Silver staining of the purified We have purified the enzyme to homogeneity by using preparation showed no additional bands. sequentially ammonium sulfate fractionation, butyl-Sepha- Downloaded by guest on September 26, 2021 Neurobiology: Wolosker et al. Proc. Natl. Acad. Sci. USA 96 (1999) 723

FIG. 2. pH and temperature dependence of racemase activity. (A) Racemase activity was assayed at 37°C in media containing 50 mM Mes-Tris (pH 6.0–6.5), 50 mM Tris⅐HCl (pH 6.8–8.8) of 50 mM CAPS-NaOH (pH 9–10.5), 20 mM L-serine, 100 ␮g͞ml purified enzyme, 1 mM EDTA, 2mMDTT,and15␮M PLP. (B) Racemase activity was performed at different temperatures in a medium containing 50 mM Tris⅐HCl, pH 8.0͞20 mM L-serine, 100 ␮g͞ml purified enzyme, 1 mM EDTA, 2 mM DTT, and 15 ␮M PLP. The reaction was stopped after 4 h and analyzed both by chemiluminescence and HPLC assay. The experiment was replicated three times by using different preparations with similar results.

rose, Q-Sepharose, mono-Q, and hydroxyapatite chromatog- enzyme activity, which can be restored by the addition of PLP raphy steps (Table 1). The overall purification from the (data not shown). AOAA and hydroxylamine, which inactivate ammonium sulfate fraction was 12,500. Enzyme activity was PLP, inhibit enzyme activity (Fig. 4). Examination of the obtained in 30% yield. Interestingly, there was an apparent absorption spectrum of the enzyme confirmed the importance increase in yield after the Q-Sepharose step, suggesting the of PLP. Thus, the normal enzyme preparation displayed removal of an inhibitor. SDS gel electrophoresis revealed a absorption peaks at 420 and 340 nm, characteristic of PLP- single band of about 37 kDa for the purified protein (Fig. 1). dependent enzymes. The peak at 420 nm, corresponding to a The enzyme was stable with no loss of activity when stored Schiff’s base complex of PLP with an active site lysine, was for 4 days at 4°C. Only modest loss of activity occurred after abolished by treatment with AOAA (Fig. 5). two cycles of freezing and thawing. The enzyme appeared to Sulfhydryl groups seem to be important for enzyme activity, be soluble with no activity detected in membrane preparations. as oxidized glutathione markedly reduced enzyme activity Activity displayed a sharp pH optimum in the alkaline range (Fig. 4). with optimal activity at pH 8–9 being about 10 times higher We wondered whether conversion of L-toD-serine might be than at pH 7 (Fig. 2). Enzyme activity was maximal at 37°C and a by-product of a different enzyme activity with nonenzymatic was abolished by boiling. racemization, giving rise to D-serine. Accordingly, we moni- Enzyme activity obeys Michaelis–Menten kinetics (Fig. 3). tored by HPLC the levels of L- and D-serine at different Monitoring the conversion of L-toD-serine, the Km was about incubation times (Table 2). Increases in formation of D-serine ␮ ͞ 10 mM with a Vmax of 5 mol mg per h. The enzyme also can are paralleled by stoichiometric decreases in levels of L-serine, convert D-toL-serine but with lesser affinity, as the Km in this making it unlikely that L-serine is converted to any other direction was 60 mM, though the Vmax was higher, at 22 compound by the enzyme. Additionally, we examined the ␮mol͞mg per h. purified enzyme for the presence of other enzyme activities Serine racemase requires PLP. Dialysis for 16 h against 1,000 that might contribute to D-serine formation indirectly. We volumes of the purification buffer without PLP abolishes found no evidence for serine:pyruvate aminotransferase ac-

FIG. 3. Kinetic parameters of racemization reaction. Initial rate of racemase activity was measured at 37°C in medium containing 50 mM Tris⅐HCl, pH 8.0, 35 ␮g͞ml purified enzyme, 1 mM EDTA, 2 mM DTT, and 15 ␮M PLP and different concentrations of either L-orD- serine. The reaction was stopped after 2 h when less than 10% substrate was consumed. Values for Km and Vmax were calculated by using the Michaelis–Menten equation. The values are representative of three experiments with different enzyme preparations. Downloaded by guest on September 26, 2021 724 Neurobiology: Wolosker et al. Proc. Natl. Acad. Sci. USA 96 (1999)

FIG. 4. Inhibition of serine racemase by PLP inhibitors and sulfhydryl oxidation. (A) Enzyme activity was monitored at 37°C in a medium containing 50 mM Tris⅐HCl, pH 8.0͞20 mM L-serine, 100 ␮g͞ml purified enzyme, 1 mM EDTA, 2 mM DTT, and 10 ␮M PLP and different concentrations of either AOAA (E) or hydroxylamine (F). (B) Reaction medium and conditions were as described in A, except that DTT was omitted from the last step of the enzyme preparation. The enzyme was preincubated for 10 min in the presence of different concentrations of oxidized glutathione (GSSG).

tivity, as L-serine levels are not altered in the presence of acids. Though a number of amino acid racemases have been pyruvate. We did not observe serine hydroxymethyltransferase purified from bacteria, no serine-specific enzyme has been activity inasmuch as we failed to detect the formation of identified previously (19–21). Serine racemase appears to be glycine after extensive incubation of the enzyme with L-serine. a very conserved enzyme. The pH optimum in the rat brain There is no evidence for serine dehydratase activity that would enzyme we have purified as well as its requirement for PLP and be associated with a marked decrease in L-serine levels. behavior in chromatographic systems resemble the activity Serine racemase is highly selective for L-serine (Table 3). characterized in crude preparations of silkworm (18). Bacterial The enzyme displays about 1.5% as much activity toward amino acid racemases display properties resembling serine L-alanine as for L-serine while no activity is demonstrated with racemase including Km values, alkaline pH optimum, and the L-threonine or L-aspartate. requirement for PLP. The alkaline pH optimum might reflect the mechanism of racemization, as PLP nonenzymatically DISCUSSION racemizes amino acids at alkaline pH (22). Serine racemase displays a Km value in the direction of L-to Our isolation of serine racemase represents the purification of D-serine resembling brain levels of L-serine and favoring the an enzyme that directly converts L-serine to D-serine. Bacteria physiologic synthesis of D-serine. Because of the much higher contain substantial levels of D-serine and many other D-amino Km value in the direction of D-toL-serine, under physiological conditions the enzyme should predominantly make D-serine. Serine racemase is a relatively small soluble protein of 37 kDa. Its absorption spectrum indicates that no minor, unde- tected protein could account for enzyme activity. Thus, the magnitude and ratios of absorption at 280, 340, and 420 nm closely resemble values for known PLP enzymes (20, 23, 24). This could be possible only if essentially all the protein is the PLP requiring racemase. D-Amino acid oxidase has been well known for many years, but its function was obscure until the demonstration of D- serine in mammalian brain. D-Amino acid oxidase is highly selective for D-serine, which thus is likely its principal, if not its sole, physiologic substrate. Our demonstration of serine race-

Table 2. Racemization of L-Serine

[L-Serine] [D-Serine] Time, h mM 0 4.00 0.0007 0.5 3.96 0.042 FIG. 5. Absorption spectra of purified serine racemase. Purified 1 3.93 0.101 enzyme (70 ␮g͞ml) was preincubated for 10 min in medium containing 4 3.65 0.342 10 mM KPi (pH 7.2), 2 mM DTT, 1 mM EDTA, and 10 ␮M PLP, either in the absence (Control) or in the presence of 1 mM AOAA. The Racemase activity was assayed at 37°C in a medium containing 50 distinct peaks of absorbance at 420 and 340 nm were not observed in mM Tris⅐HCl, pH 8.0, 4 mM L-serine, 40 ␮g͞ml purified enzyme, 1 mM the presence of buffer alone or when BSA was used instead of serine EDTA, 2 mM DTT, and 15 ␮M PLP. Samples were analyzed for amino racemase. acid emantiomers by HPLC as described. Downloaded by guest on September 26, 2021 Neurobiology: Wolosker et al. Proc. Natl. Acad. Sci. USA 96 (1999) 725

Table 3. Substrate specificity of serine racemase from Pfizer (to K.S.), and a Howard Hughes fellowship for physicians (to C.D.F.). H.W. is a Pew fellow. Specific activity, ␮ ͞ Amino acid mol mg per h % control 1. Corrigan, J. J. (1969) Science 164, 142–149. L-Serine 4.8 100 2. Corrigan, J. J. & Srinivasan, N. G. (1966) Biochemistry 5, L-Alanine 0.012 1.5 1185–1190. 3. Nagata, Y., Konno, R., Yasumura, Y. & Akino, T. (1989) L-Threonine 0 0 Biochem. J. 257, 291–292. L-Aspartate 0 0 4. Hashimoto, A., Nishikawa, T., Hayashi, T., Fujii, N., Harada, K., Racemase activity was assayed at 37°C in a medium containing 50 Oka, T. & Takahashi, K. (1992) FEBS Lett. 296, 33–36. mM Tris⅐HCl, pH 8.0, 20 mM L-amino acids, 100 ␮g͞ml purified 5. Hashimoto, A., Kumashiro, S., Nishikawa, T., Oka, T., Taka- enzyme, 1 mM EDTA, 2 mM DTT, and 15 ␮M PLP. After 8 h, the hashi, K., Mito, T., Takashima, S., Doi, N., Mizutani, Y., reaction was terminated by the addition of 5% TCA, and samples were Yamazaki, T., Kaneko, T. & Ootomo, E. (1993) J. Neurochem. 61, analyzed by HPLC as described. The data represent a typical exper- 783–786. iment that was replicated three times by using different preparations 6. Nagata, Y., Horiike, K. & Maeda, T. (1994) Brain Res. 634, with similar results. 291–295. 7. Dunlop, D. S., Neidle, A., McHale, D., Dunlop, D. M. & Lajtha, mase now establishes that both biosynthetic and degrading A. (1986) Biochem. Biophys. Res. Commun. 141, 27–32. enzymes for D-serine exist in mammalian brain. 8. Hashimoto, A., Oka, T. & Nishikawa, T. (1995) Eur. J. Neurosci. Accumulating evidence establishes that D-serine is the pre- 7, 1657–1663. dominant endogenous ligand for the ‘‘glycine site’’ of the 9. Schell, M. J., Cooper, O. B. & Snyder, S. H. (1995) Proc. Natl. NMDA receptor. Its localization in most brain areas resemble Acad. Sci. USA 94, 2013–2018. 10. Schell, M. J., Molliver, M. E. & Snyder, S. H. (1995) Proc. Natl. NMDA receptor distribution more closely than glycine (10, Acad. Sci. USA 92, 3948–3952. 11). It is released from type II astrocytes by glutamatergic 11. Schell, M. J., Brady, R. O., Jr., Molliver, M. E. & Snyder, S. H. stimulation (10), and it is at least as active as glycine at the (1997) J. Neurosci. 17, 1604–1615. ‘‘glycine site’’ (14). Moreover, recently, we have shown that 12. Johnson, J. W. & Ascher, P. (1987) Nature (London) 325, endogenous D-serine is required for physiologic NMDA neu- 529–531. rotransmission. Thus, treatment of brain slices or cultures with 13. Kleckner, N. W. & Dingledine, R. (1988) Science 241, 835–837. D-amino acid oxidase, under conditions in which D-serine is 14. Matsui, T., Sekiguchi, M., Hashimoto, A., Tomita, U., Nishikawa, completely degraded, greatly reduces NMDA transmission T. & Wada, K. (1995) J. Neurochem. 65, 454–458. measured electrophysiologically or biochemically in terms of 15. Dunlop, D. S. & Neidle, A. (1997) Biochem. Biophys. Res. Commun. 235, 26–30. stimulation of nitric oxide synthase activity and levels of cyclic 16. Scannone, H., Wellner, D. & Novogrodsky, A. (1964) Biochem- GMP (J. P.M., A. Parent, D. Linden, H.W., C.D.F., R.O.B., istry 11, 1742–1745. M. A. Rogawski, and S.H.S., unpublished data). 17. Hashimoto, A., Nishikawa, T., Oka, T., Takahashi, K. & Hayashi, We have obtained amino acid sequence for mammalian T. (1992) J. Chromatogr. 582, 41–48. serine racemase, reflecting a protein with no major similarity 18. Uo, T., Yoshimura, T., Shimizu, S. & Esaki, N. (1998) Biochem. to any other known protein (H.W., S. Blackshaw, and S.H.S., Biophys. Res. Commun. 246, 31–34. unpublished data). Targeted deletion of this enzyme protein 19. Wood, W. A. & Gunsalus, I. C. (1951) J. Biol. Chem. 190, may enhance substantially our understanding of NMDA neu- 403–416. rotransmission. Drugs inhibiting serine racemase may provide 20. Yorifuji. T., Misono, H. & Soda, K. (1971) J. Biol. Chem. 246, a useful therapeutic approach in disease entities for which 5093–5101. 21. Svensson, M. L. & Gatenbeck, S. (1981) Arch. Microbiol. 129, NMDA receptor antagonists have displayed efficacy, such as 213–215. in the treatment of stroke and other forms of overexcitation in 22. Olivard, J., Metzler, D. E. & Snell, E. E. (1952) J. Biol. Chem. 191, the brain. 669–674. 23. Manohar, R., Apu Rao, A. G. & Appaji Rao, N. (1984) Bio- This work was supported by U.S. Public Health Service Grant chemistry 23, 4116–4122. MH-18501, the Theodore and Vada Stanley Foundation, and Re- 24. Ishikawa, K., Kaneko, E. & Ichiyama, A. (1996) J. Biochem. 119, search Scientist Award DA00074 (to S.H.S.), a summer fellowship 970–978. Downloaded by guest on September 26, 2021