title: Nitric oxide trapping of tyrosyl radicals generated during prostaglandin endoperoxide synthase turnover. Detection of the radical derivative of tyrosine 385.
abstract: Tyrosyl radicals have been detected during turnover of prostaglandin endoperoxide H synthase (PGHS), and they are speculated to participate in cyclooxygenase catalysis. Spectroscopic approaches to elucidate the identity of the radicals have not been definitive, so we have attempted to trap the radical(s) with nitric oxide (NO). NO quenched the EPR signal generated by reaction of purified ram seminal vesicle PGHS with arachidonic acid, suggesting that NO coupled with a tyrosyl radical to form inter alia nitrosocyclohexadienone. Subsequent formation of nitrotyrosine was detected by Western blotting of PGHS incubated with NO and arachidonic acid or organic hydroperoxides using an antibody against nitrotyrosine. Both arachidonic acid and NO were required to form nitrotyrosine, and tyrosine nitration was blocked by the PGHS inhibitor indomethacin. The presence of superoxide dismutase had no effect on nitration, indicating that peroxynitrite was not the nitrating agent. To identify which tyrosines were nitrated, PGHS was digested with trypsin, and the resulting peptides were separated by high pressure liquid chromatography and monitored with a diode array detector. A single peptide was detected that exhibited a spectrum consistent with the presence of nitrotyrosine. Consistent with Western blotting results, both NO and arachidonic acid were required to observe nitration of this peptide, and its formation was blocked by the PGHS inhibitor indomethacin. Peptide sequencing indicated that the modified residue was tyrosine 385, the source of the putative catalytically active tyrosyl radical.
authors.affiliation: Department of Biochemistry, A.B. Hancock, Jr. Memorial Laboratory for Cancer Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: D C
authors.initials: DC
authors.lastname: Goodwin
authors.forename: M R
authors.initials: MR
authors.lastname: Gunther
authors.forename: L C
authors.initials: LC
authors.lastname: Hsi
authors.forename: B C
authors.initials: BC
authors.lastname: Crews
authors.forename: T E
authors.initials: TE
authors.lastname: Eling
authors.forename: R P
authors.initials: RP
authors.lastname: Mason
authors.forename: L J
authors.initials: LJ
authors.lastname: Marnett
mesh_terms: D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000378:metabolism; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism; D007213:Indomethacin / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000378:metabolism; D013774:Tetranitromethane / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism*
chemical_list: D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D005609:Free Radicals; D007527:Isoenzymes; D010446:Peptide Fragments; D016718:Arachidonic Acid; D009569:Nitric Oxide; D014443:Tyrosine; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D013774:Tetranitromethane; D007213:Indomethacin