Prostaglandin hydroperoxidase, an integral part of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes.

Ohki, S; Ogino, Nobuchika; Yamamoto, Shozo; Hayaishi, Osamu

doi:10.1016/s0021-9258(17)37880-8

Cited by 344 publications

(28 citation statements)

References 24 publications

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“…The standard lipoxygenase reaction mixture contained 0.1 M Tris-HCl (pH 8.0), 1 mM Dtryptophan, and 60 µM arachidonate at 23 °C. Peroxidase activity was measured spectrophotometrically at 436 nm (Ohki et al, 1979) in a reaction mixture containing 0.1 M Tris-HCl (pH 8.0), 0.5 mM guaiacol, 0.4 mM hydrogen peroxide, and 1 µM heme. One unit of peroxidase produces a velocity of 1 nmol of hydrogen peroxide/min.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Hydroperoxide-Induced Tyrosyl Radicals and Lipoxygenase Activity in Aspirin-Treated Human Prostaglandin H Synthase-2

et al. 1997

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A hydroperoxide-induced tyrosyl radical has been proposed as a key cyclooxygenase intermediate for the "basal" isoform of prostaglandin H synthase (PGHS-1). In the present study with the "inducible" isoform (PGHS-2), hydroperoxide was also found to generate a radical in high yield, a wide singlet at g = 2.0058 (29 G peak to trough). Reaction of PGHS-2 with a tyrosine-modifying reagent, tetranitromethane (TNM), resulted in cyclooxygenase inactivation and a much narrower radical EPR signal (22 G peak to trough). Addition of a cyclooxygenase inhibitor, nimesulide, similarly resulted in a narrow PGHS-2 radical. In PGHS-1, cyclooxygenase inhibition by tyrosine nitration with TNM or by active site ligands leads to generation of a narrow EPR instead of a wide EPR, with both signals originating from authentic tyrosyl radicals, indicating that the hydroperoxide-induced radicals in PGHS-2 are also tyrosyl radicals. Treatment of PGHS-2 with aspirin (acetyl salicylic acid, ASA) was previously shown to result in acetylation of a specific serine residue, cyclooxygenase inhibition, and increased lipoxygenase activity. Acetylation of PGHS-1 by ASA, in contrast, inhibited both lipoxygenase and cyclooxygenase activity. We now have found the ASA-treated PGHS-2 radical to be indistinguishable from that in control PGHS-2. Addition of nimesulide to ASA-treated PGHS-2 inhibited the lipoxygenase and resulted in a narrow radical EPR like that seen in PGHS-2 treated with TNM or nimesulide alone. Retention of PGHS-2 oxygenase activity was thus associated with retention of the native radical, and loss of activity was associated with alteration of the radical. Both native and ASA-treated PGHS-2 produced only the R stereoisomer of 11- and 15-HETE, demonstrating that the lipoxygenase stereochemistry was not changed by ASA. Native and ASA-treated PGHS-2 had lipoxygenase K(m) values considerably higher than that of the control PGHS-2 cyclooxygenase. Taken together, these results suggest that the same PGHS-2 tyrosyl radical serves as the oxidant for both cyclooxygenase and lipoxygenase catalysis and that acetylation of PGHS-2 by ASA favors arachidonate binding in an altered conformation which results in abstraction of the pro-R hydrogen from C13 and formation of 11(R)- and 15(R)-HETE.

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Section: Methodsmentioning

confidence: 99%

Analysis of Hydroperoxide-Induced Tyrosyl Radicals and Lipoxygenase Activity in Aspirin-Treated Human Prostaglandin H Synthase-2

et al. 1997

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“…PBZ is a well-studied anti-inflammatory agent. Early studies found that it suppressed the release of lysosomal enzymes which was believed to play a role in joint disease, but PBZ is best known for its ability to inhibit prostaglandin synthesis from arachidonic acid. , It is well-known that prostaglandin H synthase (PHS) plays a pivotal role in the synthesis of hydroxy endoperoxide (PGH 2 ), which serves as a precursor to many other important biomolecules. − It has been shown that the hydroperoxidase activity of PHS can also oxidize PBZ to initially generate a carbon-centered radical that reacts with molecular oxygen to form a peroxyl radical and that the latter radical, per se, is required to inactivate the cyclooxygenase activity of PHS…”

Section: Introductionmentioning

confidence: 99%

Phenylbutazone Oxidation via Cu,Zn-SOD Peroxidase Activity: An EPR Study

Aljuhani

Whittal²,

Khan³

et al. 2015

Chem. Res. Toxicol.

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We investigated the effect of Cu,Zn-superoxide dismutase (Cu,Zn-SOD)-peroxidase activity on the oxidation of the nonsteroidal anti-inflammatory drug phenylbutazone (PBZ). We utilized electron paramagnetic resonance (EPR) spectroscopy to detect free radical intermediates of PBZ, UV-vis spectrophotometry to monitor PBZ oxidation, oxygen analysis to determine the involvement of C-centered radicals, and LC/MS to determine the resulting metabolites. Using EPR spectroscopy and spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we found that the spin adduct of CO3(•-) (DMPO/(•)OH) was attenuated with increasing PBZ concentrations. The resulting PBZ radical, which was assigned as a carbon-centered radical based on computer simulation of hyperfine splitting constants, was trapped by both DMPO and MNP spin traps. Similar to Cu,Zn-SOD-peroxidase activity, an identical PBZ carbon-centered radical was also detected with the presence of both myeloperoxidase (MPO/H2O2) and horseradish peroxidase (HRP/H2O2). Oxygen analysis revealed depletion in oxygen levels when PBZ was oxidized by SOD peroxidase-activity, further supporting carbon radical formation. In addition, UV-vis spectra showed that the λmax for PBZ (λ = 260 nm) declined in intensity and shifted to a new peak that was similar to the spectrum for 4-hydroxy-PBZ when oxidized by Cu,Zn-SOD-peroxidase activity. LC/MS evidence supported the formation of 4-hydroxy-PBZ when compared to that of a standard, and 4-hydroperoxy-PBZ was also detected in significant yield. These findings together indicate that the carbonate radical, a product of SOD peroxidase activity, appears to play a role in PBZ metabolism. Interestingly, these results are similar to findings from heme peroxidase enzymes, and the context of this metabolic pathway is discussed in terms of a mechanism for PBZ-induced toxicity.

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“…In the enzyme assay, 14 C-labeled PGH 1 , an unstable endoperoxide, could be isolated by performing thin-layer chromatography at −25°C. Further purification of the oxygenase was achieved by isoelectric focusing (5,6). The purified enzyme was found to have dual functions; namely, the catalyzation of both the bis-oxygenation of 8, 11, 14-eicosatrienoic acid to form PGG 1 (fatty acid cyclooxygenase activity) and the reduction of the 15-hydroperoxy group of PGG 1 to a 15-hydroxyl group, leading to the formation of PGH 1 (PG hydroperoxidase activity).…”

mentioning

confidence: 99%