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Preparation and Assay of Recombinant Serine Racemase

  • Florian Baumgart
  • Clara Aicart-Ramos
  • Ignacio Rodriguez-CrespoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 794)

Abstract

Serine racemase is a glial and neuronal enzyme that reversibly converts l-serine to d-serine, an endogenous co-agonist of N-methyl-d-aspartate receptor type glutamate receptors (NMDARs). Here we present methods to recombinantly express and purify serine racemase in bacteria and two complementary ways to determine d-serine levels in unknown samples. Furthermore, a detailed protocol of serine racemase activity assays is described that can be used to screen for activators and inhibitors in vitro.

Key words

Serine racemase d-Serine PLP enzymes Racemization 

Notes

Acknowledgments

This work was supported by Grants BFU2006-05395 and BFU2009-10442 from the Spanish MICINN.

References

  1. 1.
    Wolosker H, Blackshaw S, Snyder S H (1999) Serine racemase: a glial enzyme synthesizing D-serine to regulate glutamate-N-methyl-D-aspartate neurotransmission. Proc Natl Acad Sci USA 96, 13409–13414.Google Scholar
  2. 2.
    Wolosker H, Sheth K N, Takahashi M et al. (1999) Purification of serine racemase: biosynthesis of the neuromodulator D-serine. Proc Natl Acad Sci USA 96, 721–725.Google Scholar
  3. 3.
    Baumgart F, Rodriguez-Crespo I (2008) D-amino acids in the brain: the biochemistry of brain serine racemase. FEBS J 275, 3538–3545.Google Scholar
  4. 4.
    Foltyn V N, Bendikov I, De Miranda J et al. (2005) Serine racemase modulates intracellular D-serine levels through an alpha,beta-elimination activity. J Biol Chem 280, 1754–1763.Google Scholar
  5. 5.
    De Miranda J, Santoro A, Engelender S, Wolosker H (2000)Human serine racemase: moleular cloning, genomic organization and functional analysis. Gene 256, 183–188.Google Scholar
  6. 6.
    Cook S P, Galve-Roperh I, Martinez del Pozo A, Rodriguez-Crespo I (2002) Direct calcium binding results in activation of brain serine racemase. J Biol Chem 277, 27782–27792.Google Scholar
  7. 7.
    Stevens E R, Esguerra M, Kim PM et al. (2003) D-serine and serine racemase are present in the vertebrate retina and contribute to the physiological activation of NMDA receptors. Proc Natl Acad Sci USA 100, 6789–6794.Google Scholar
  8. 8.
    De Miranda J, Panizzutti R, Foltyn V N, Wolosker H (2002) Cofactors of serine racemase that physiologically stimulate the synthesis of the N-methyl-D-aspartate (NMDA) receptor coagonist D-serine. Proc Natl Acad Sci USA 99, 14542–14547.Google Scholar
  9. 9.
    Baumgart F, Mancheno J M, Rodriguez-Crespo I (2007) Insights into the activation of brain serine racemase by the multi-PDZ domain glutamate receptor interacting protein, divalent cations and ATP. FEBS J 274, 4561–4571.Google Scholar
  10. 10.
    Kim P M, Aizawa H, Kim PS et al. (2005) Serine racemase: activation by glutamate neurotransmission via glutamate receptor interacting protein and mediation of neuronal migration. Proc Natl Acad Sci USA 102, 2105–2110.Google Scholar
  11. 11.
    Smith M A, Mack V, Ebneth A et al. (2010) The structure of mammalian serine racemase: evidence for conformational changes upon inhibitor binding. J Biol Chem 285, 12873–12881.Google Scholar
  12. 12.
    Martinez del Pozo A, Merola M, Ueno H et al. (1989) Stereospecificity of reactions catalyzed by bacterial D-amino acid transaminase. J Biol Chem 264, 17784–17789.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Florian Baumgart
    • 1
  • Clara Aicart-Ramos
    • 1
  • Ignacio Rodriguez-Crespo
    • 1
    Email author
  1. 1.Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias QuímicasUniversidad ComplutenseMadridSpain

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