Abstract
d-Serine is a transmitter-like molecule that physiologically activates NMDA receptors in the brain. Although d-serine was thought to be exclusively released by astrocytes, we recently demonstrated endogenous d-serine release from neurons in cultures and slices. So far high-performance liquid chromatography (HPLC) has been the standard technique to monitor d-serine and other amino acids. This method employs pre-column derivatization with a chiral reagent to produce fluorescence derivatives that can be further separated on a reversed-phase column. Due to the close retention times of l-serine, l-glutamine, and d-serine, the quantification of low levels of endogenous d-serine synthesis and release from cell cultures and tissues can be challenging. We here describe an enzymatic treatment method to specifically destroy l-glutamine and l-serine by glutaminase and l-serine dehydratase enzymes, respectively, allowing accurate determination of nanomolar d-serine concentrations by subsequent HPLC analysis.
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References
Basu A C et al. (2009) Targeted disruption of serine racemase affects glutamatergic neurotransmission and behavior. Mol Psychiatry 14, 719–727.
Mothet J P et al. (2000) D-serine is an endogenous ligand for the glycine site of the N-methyl-D- aspartate receptor. Proc Natl Acad Sci USA 97, 4926–4931.
Wolosker H et al. (2008) D-amino acids in the brain: D-serine in neurotransmission and neurodegeneration. FEBS J 275, 3514–3526.
Lipton S A (2006) Paradigm shift in neuroprotection by NMDA receptor blockade: memantine and beyond. Nature Reviews 5, 160–170.
Shleper M, Kartvelishvily E, Wolosker H (2005) D-serine is the dominant endogenous coagonist for NMDA receptor neurotoxicity in organotypic hippocampal slices. J Neurosci 25, 9413–9417.
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.
Hashimoto A et al. (1992) Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid chromatography after derivatization with N-tert.-butyloxycarbonyl-L-cysteine and o-phthaldialdehyde. J Chromatogr 582, 41–48.
Foltyn V N, Wolosker H (2006) Recombinant serine racemase preparation. In: Fisher G H, Brueckner N, Fujii H, Homma H, Konno R (eds), D-Amino acids: A new frontier in amino acid and protein research, Nova Science Publishers Inc., pp 463–466.
Price P J, Brewer G J (2001) Serum-free media for neural cell cultures. In: Protocols for neural cell culture, Human Press, Totowa, New Jersey, pp 255–264.
Kartvelishvily E et al. (2006) Neuron-derived D-serine release provides a novel means to activate N-methyl-D-aspartate receptors. J Biol Chem 281, 14151–14162.
Rosenberg D et al. (2010) Neuronal release of D-serine: a physiological pathway controlling extracellular D-serine concentration. FASEB J 24, 2951–2961.
Balan L et al. (2009) Feedback inactivation of D-serine synthesis by NMDA receptor-elicited translocation of serine racemase to the membrane. Proc Natl Acad Sci USA 106, 7589–7594.
Foltyn V N et al. (2005) Serine racemase modulates intracellular D-serine levels through an alpha,beta-elimination activity. J Biol Chem 280, 1754–1763.
Ogawa H et al. (2006) Enzymatic and biochemical properties of a novel human serine dehydratase isoform. Biochim Biophys Acta 1764, 961–971.
Ito T et al. (2008) A novel zinc-dependent D-serine dehydratase from Saccharomyces cerevisiae. Biochem J 409, 399–406.
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Radzishevsky, I., Wolosker, H. (2012). An Enzymatic-HPLC Assay to Monitor Endogenous d-Serine Release from Neuronal Cultures. In: Pollegioni, L., Servi, S. (eds) Unnatural Amino Acids. Methods in Molecular Biology, vol 794. Humana Press. https://doi.org/10.1007/978-1-61779-331-8_19
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DOI: https://doi.org/10.1007/978-1-61779-331-8_19
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