title: Cross-linking of serine racemase dimer by reactive oxygen species and reactive nitrogen species.
abstract: Serine racemase (SR) is the only identified enzyme in mammals responsible for isomerization of L-serine to D-serine, a coagonist at N-methyl-D-aspartate (NMDA) receptors in the forebrain. Our previous data showed that an apparent SR dimer resistant to sodium dodecyl sulfate and β-mercaptoethanol was elevated in microglial cells after proinflammatory activation. Because the activation of microglia is typically associated with an oxidative burst, oxidative cross-linking between SR subunits was speculated. In this study, an siRNA technique was employed to confirm the identity of this SR dimer band. The oxidative species potentially responsible for the cross-linking was investigated with recombinant SR protein. The data indicate that nitric oxide, peroxynitrite, and hydroxyl radical were the likely candidates, whereas superoxide and hydrogen peroxide per se failed to contribute. Furthermore, the mechanism of formation of SR dimer by peroxynitrite oxidation was studied by mass spectrometry. A disulfide bond between Cys₆ and Cys₁₁₃ was identified in 3-morpholinosydnonimine hydrochloride (SIN-1)-treated SR monomer and dimer. Activity assays indicated that SIN-1 treatment decreased SR activity, confirming our previous conclusion that noncovalent dimer is the most active form of SR. These findings suggest a compensatory feedback in which the consequences of neuroinflammation might dampen D-serine production to limit excitotoxic stimulation of NMDA receptors.
authors.affiliation: Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Steven W
authors.initials: SW
authors.lastname: Barger
mesh_terms: D015097:3-Mercaptopropionic Acid / Q000494:pharmacology; D000596:Amino Acids / Q000378:metabolism; D002461:Cell Line, Transformed; D002614:Chelating Agents / Q000494:pharmacology; D004347:Drug Interactions; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017665:Hydroxyl Radical / Q000494:pharmacology; D019271:Hypoxanthine / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D013058:Mass Spectrometry; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D010417:Pentaerythritol Tetranitrate / Q000031:analogs & derivatives / Q000494:pharmacology; D004369:Pentetic Acid / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000494:pharmacology; D034622:RNA Interference / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology; D019998:Racemases and Epimerases / Q000187:drug effects / Q000378:metabolism*; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013096:Spermine / Q000031:analogs & derivatives / Q000494:pharmacology; D000099300:Serine Racemase
chemical_list: D015097:3-Mercaptopropionic Acid; D000596:Amino Acids; D002614:Chelating Agents; D006861:Hydrogen Peroxide; D017665:Hydroxyl Radical; D019271:Hypoxanthine; D008070:Lipopolysaccharides; D020030:Nitric Oxide Donors; D016877:Oxidants; D010417:Pentaerythritol Tetranitrate; D004369:Pentetic Acid; D030421:Peroxynitrous Acid; D034741:RNA, Small Interfering; D019998:Racemases and Epimerases; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D012967:Sodium Dodecyl Sulfate; D013096:Spermine; D000099300:Serine Racemase; C091861:spermine nitric oxide complex; C100210:pentrinitrol