Glutamate is the major excitatory neurotransmitter in the central nervous system, and many of its effects are mediated by N-methyl-D-aspartate receptors (NMDARs). Malfunction of NMDARs may participate in many neurodegenerative and psychiatric disorders. Accumulating evidence shows that D-serine physiologically regulates the activity of NMDARs, and serine racemase (SR) is the main enzyme in mammals responsible for endogenous production of D-serine. To analyze structure-function relationships of this enzyme, we cloned the cDNA of human SR, expressed it in E. coli and purified it with affinity chromatography. Michaelis-Menten kinetics indicated a Km value of 14 mM and Vmax value of 3.66 μmol·mg-1·hr-1 at 37°C when L-serine was used as a substrate for purified SR. Native polyacrylamide gel electrophoresis, gel-filtration chromatography and protein cross-linking experiments revealed that dimer is the major form of recombinant SR in phosphate buffer, though there were small amounts of monomer, tetramer, and larger aggregates. Activity assays showed that the dimeric gel-filtration fraction held the highest activity. Addition of dithiothreitol (DU) in buffers increased the activity of SR through enhancing the recovery of noncovalent SR dimer, suggesting that optimal activity depends on prevention of disulfide-bound dimer, tetramer, and aggregates during refolding and/or reduction of the sulfhydryl group of critical cysteine residue(s) located in the enzyme's active center or modulatory site. Data also showed that lipopolysaccharide (LPS) elevated the sodium dodecyl sulphate (SDS)/Rmercaptoethanol (pME)-resistant SR dimer in microglia. A peroxynitrite donor: 3-morpholinosydnonimine hydrochloride (SIN-1) treatment of recombinant human SR increased SDS/βME-resistant dimer and decreased enzyme activity, which suggests that peroxynitrite may be responsible for the elevation of SDS/βME-resistant SR dimer in microglia under LPS stimulation. Mass spectroscopy revealed that a disulfide bond is formed between Cys6 and Cys113 intramolecularly and/or intermolecularly in SDS/βME-resistant SR dimer, which indicates that Cys6 and/or Cys113 are critical for SR structure and catalysis. The above evidence leads to the following conclusion: noncovalent SR dimer with free sulfhydryl group(s) is essential for full enzyme activity.
Further, phosphorylation of SR by protein kinase PKA and PKC, the effects of glutamate and cannabinoid receptor on D-serine production in glia, and SR expression during NTera2 cell differentiation were investigated.
|Advisor:||Barger, Steven W.|
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 71/06, Dissertation Abstracts International|
|Subjects:||Molecular biology, Neurosciences|
|Keywords:||D-serine, NMDA receptor, Neurotransmitters, Serine racemase|
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