共查询到20条相似文献,搜索用时 15 毫秒
1.
J Schreiber G L Foureman M F Hughes R P Mason T E Eling 《The Journal of biological chemistry》1989,264(14):7936-7943
We report here the application of the electron spin resonance technique to detect free radicals formed by the hydroperoxidase activity of prostaglandin H synthase in cells. Studies were done using keratinocytes obtained from hairless mice. These cells can be prepared in large number and possess significant prostaglandin H synthase activity. Initial attempts to directly detect free radical metabolites of several amines in cells were unsuccessful. A technique was developed based on the ability of some free radicals formed by prostaglandin hydroperoxidase to oxidize reduced glutathione (GSH) to a thiyl radical, which was trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Phenol and aminopyrine are excellent hydroperoxidase substrates for this purpose and thus were used for all further experiments. Using this approach we detected the DMPO/GS.thiyl radical adduct catalyzed by cellular prostaglandin hydroperoxidase. The formation of the radical was dependent on the addition of substrate, inhibited by indomethacin, and supported by either exogenous arachidonic acid or endogenous arachidonic acid released from phospholipid stores by Ca2+ ionophore A-23187. The addition of GSH significantly increased the intracellular GSH concentration and concomitantly stimulated the formation of the DMPO/GS.thiyl radical adduct. Phenol, but not aminopyrine, enhanced thiyl radical adduct formation and prostaglandin formation with keratinocytes while both cofactors were equally effective in incubations containing microsomes prepared from keratinocytes. These results suggest that prostaglandin hydroperoxidase-dependent co-oxidation of chemicals can result in the intracellular formation of free radical metabolites. 相似文献
2.
R J Kulmacz 《Biochemical and biophysical research communications》1987,148(2):539-545
Prostaglandin H synthase was incubated with [14C]arachidonate and then analyzed by polyacrylamide gel electrophoresis under denaturing conditions and by high pressure liquid chromatography. A maximum of 1 mol of arachidonate metabolite was found to become attached per mol of synthase subunit in a time-dependent process that was much slower than the rate of self-catalyzed inactivation of the cyclooxygenase activity. Incubation of a mixture of the synthase and ovalbumin with [14C]arachidonate resulted in a selective attachment of radiolabel to the synthase. These results suggest the presence of a single site on the synthase that is susceptible to reaction with an arachidonate metabolite. 相似文献
3.
The oxidizing power of the glutathione thiyl radical as measured by its electrode potential at physiological pH 总被引:1,自引:0,他引:1
The oxidizing power of the thiyl radical (GS*) produced on oxidation of glutathione (GSH) was determined as the mid-point electrode potential (reduction potential) of the one-electron couple E(m)(GS*,H+/GSH) in water, as a function of pH over the physiological range. The method involved measuring the equilibrium constants for electron-transfer equilibria with aniline or phenothiazine redox indicators of known electrode potential. Thiyl and indicator radicals were generated in microseconds by pulse radiolysis, and the position of equilibrium measured by fast kinetic spectrophotometry. The electrode potential E(m)(GS*,H+/GSH) showed the expected decrease by approximately 0.06 V/pH as pH was increased from approximately 6 to 8, reflecting thiol/thiolate dissociation and yielding a value of the reduction potential of GS*=0.92+/-0.03 V at pH 7.4. An apparently almost invariant potential between pH approximately 3 and 6, with potentials significantly lower than expected, is ascribed at least in part to errors arising from radical decay during the approach to the redox equilibrium and slow electron transfer of thiol compared to thiolate. 相似文献
4.
The metabolism of styrene by prostaglandin hydroperoxidase and horseradish peroxidase was examined. Ram seminal vesicle microsomes in the presence of arachidonic acid or hydrogen peroxide and glutathione converted styrene to glutathione adducts. Neither styrene 7,8-oxide nor styrene glycol was detected as a product in the incubation. Also, the addition of styrene 7,8-oxide and glutathione to ram seminal vesicle microsomes did not yield styrene glutathione adducts. The peroxidase-generated styrene glutathione adducts were isolated by high pressure liquid chromatography and characterized by NMR and tandem mass spectrometry as a mixture of (2R)- and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione. (1R)- and (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione were not formed by the peroxidase system. The addition of phenol or aminopyrine to incubations, which greatly enhances the oxidation of glutathione to a thiyl radical by peroxidases, increased the formation of styrene glutathione adducts. We propose a new mechanism for the formation of glutathione adducts that is independent of epoxide formation but dependent on the initial oxidation of glutathione to a thiyl radical by the peroxidase, and the subsequent reaction of the thiyl radical with a suitable substrate, such as styrene. 相似文献
5.
B H Stock J Schreiber C Guenat R P Mason J R Bend T E Eling 《The Journal of biological chemistry》1986,261(34):15915-15922
We have proposed, using styrene as a model, a new mechanism for the formation of glutathione conjugates that is independent of epoxide formation but dependent on the oxidation of glutathione to a thiyl radical by peroxidases such as prostaglandin H synthase or horseradish peroxidase. The thiyl radical reacts with styrene to yield a carbon-centered radical which subsequently reacts with molecular oxygen to give the styrene-glutathione conjugate. We have used electron spin resonance spin trapping techniques to detect the proposed free radical intermediates. A styrene carbon-centered radical was trapped using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and t-nitrosobutane. The position of the carbon-centered radical was confirmed to be at carbon 7 by the use of specific 2H-labeled styrenes. The addition of the spin trap DMPO inhibited both the utilization of molecular oxygen and the formation of styrene-glutathione conjugates. Under anaerobic conditions additional styrene-glutathione conjugates were formed, one of which was identified by fast atom bombardment mass spectrometry as S-(2-phenyl)ethylglutathione. The glutathione thiyl radical intermediate was observed by spin trapping with DMPO. These results support the proposed free radical-mediated formation of styrene-glutathione conjugates by peroxidase enzymes. 相似文献
6.
The thiyl free radical is formed in the L-cysteine/N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) system (pH 7.8) without exposure to light as detected by the ESR spin trapping technique. The formation of N-methyl-N'-nitroguanidine in the system is identified by thin-layer chromatography. The hypothesis that the thiyl radical is formed by the attack of the nucleophilic reagent L-cysteine on the nitroso group of MNNG is verified by these results. 相似文献
7.
D Ross R J Mehlhorn P Moldeus M T Smith 《The Journal of biological chemistry》1985,260(30):16210-16214
Diethylstilbestrol is carcinogenic in rodents and in humans and its peroxidatic oxidation in utero has been associated with its carcinogenic activity. Horseradish peroxidase-catalyzed oxidation of [14C]diethylstilbestrol and [14C]diethylstilbestrol analogs induced binding of radiolabel to DNA only when the compound contained a free hydroxy group (Metzler, M., and Epe, B. (1984) Chem. Biol. Interact. 50, 351-360). We have found that horseradish peroxidase or prostaglandin-H synthase-catalyzed oxidation of diethylstilbestrol in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide caused the generation of an ESR signal indicative of a free radical intermediate (aN = 14.9 G, aH = 18.3 G). The identity of the trapped radical could not be identified on the basis of published hyperfine coupling constants, but the observation that horseradish peroxidase-catalyzed oxidation of 1-naphthol produced an identical ESR signal suggests that the radical was either a phenoxy or phenoxy-derived radical. During horseradish peroxidase-catalyzed oxidation of diethylstilbestrol in the presence of glutathione the thiol reduced the diethylstilbestrol radical to generate a thiyl radical. This was shown by a thiol-dependent oxygen uptake during horseradish peroxidase-catalyzed oxidation of diethylstilbestrol and the observation of an ESR signal consistent with 5,5-dimethylpyrroline-N-oxide-glutathionyl radical adduct formation. A diethylstilbestrol analog devoid of free hydroxy groups, namely diethylstilbestrol dipropionate, did not produce an ESR signal above control levels during horseradish peroxidase-catalyzed metabolism in the presence of 5,5-dimethylpyrroline-N-oxide. Thus, free radicals are formed during peroxidatic oxidation of diethylstilbestrol and must be considered as possible determinants of the genotoxic activity of this compound. 相似文献
8.
C M Markey A Alward P E Weller L J Marnett 《The Journal of biological chemistry》1987,262(13):6266-6279
The peroxidase activity of prostaglandin H (PGH) synthase catalyzes reduction of 5-phenyl-4-pentenyl hydroperoxide to 5-phenyl-4-pentenyl alcohol with a turnover number of approximately 8000 mol of 5-phenyl-4-pentenyl hydroperoxide/mol of enzyme/min. The kinetics and products of reaction establish PGH synthase as a classical heme peroxidase with catalytic efficiency similar to horseradish peroxidase. This suggests that the protein of PGH synthase evolved to facilitate peroxide heterolysis by the heme prosthetic group. Comparison of an extensive series of phenols, aromatic amines, beta-dicarbonyls, naturally occurring compounds, and nonsteroidal anti-inflammatory drugs indicates that considerable differences exist in their ability to act as reducing substrates. No correlation is observed between the ability of compounds to support peroxidatic hydroperoxide reduction and to inhibit cyclooxygenase. In addition, the resolved enantiomers of MK-410 and etodolac exhibit dramatic enantiospecific differences in their ability to inhibit cyclooxygenase but are equally potent as peroxidase-reducing substrates. This suggests that there are significant differences in the orientation of compounds at cyclooxygenase inhibitory sites and the peroxidase oxidation site(s). Comparison of 5-phenyl-4-pentenyl hydroperoxide reduction by PGH synthase and horseradish peroxidase reveals considerable differences in reducing substrate specificity. Both the cyclooxygenase and peroxidase activities of PGH synthase inactivate in the presence of low micromolar amounts of hydroperoxides and arachidonic acid. PGH synthase was most sensitive to arachidonic acid, which exhibited an I50 of 0.6 microM in the absence of all protective agents. Inactivation by hydroperoxides requires peroxidase turnover and can be prevented by reducing substrates. The I50 values for inactivation by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid are 4.0 and 92 microM, respectively, in the absence and presence of 500 microM phenol, a moderately good reducing substrate. The ability of compounds to protect against hydroperoxide-induced inactivation correlates directly with their ability to act as reducing substrates. Hydroquinone, an excellent reducing substrate, protected against hydroperoxide-induced inactivation when present in less than 3-fold molar excess over hydroperoxide. The presence of a highly efficient hydroperoxide-reducing activity appears absolutely essential for protection of the cyclooxygenase capacity of PGH synthase. The peroxidase activity is, therefore, a twin-edged sword, responsible for and protective against hydroperoxide-dependent inactivation of PGH synthase.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
9.
Prostaglandin H synthase (PGHS) is an autocatalytic enzyme which plays a key role in the arachidonic acid metabolic pathway.
PGHS mediates the formation of prostaglandin H2, the precursor for a number of prostaglandins which are important in a wide variety of biological processes, including inflammation,
blood clotting, renal function, and tumorigenesis. Here we present a Michaelis-Menten-style model for PGHS. A stability analysis
determines when the reaction becomes self-sustaining, and can help explain the regulation of PGHS activity in vivo. We also
consider a quasi-steady-state approximation (QSSA) for the model, and present conditions under which the QSSA is expected
to be a good approximation. Applying the QSSA for this model can be useful in computationally intensive modeling endeavors
involving PGHS. 相似文献
10.
The formation of aminopyrine cation radical by the peroxidase activity of prostaglandin H synthase and subsequent reactions of the radical 总被引:2,自引:0,他引:2
The oxidation of aminopyrine to an aminopyrine cation radical was investigated using a solubilized microsomal preparation or prostaglandin H synthase purified from ram seminal vesicles. Aminopyrine was oxidized to an aminopyrine cation radical in the presence of arachidonic acid, hydrogen peroxide, t-butyl hydroperoxide or 15-hydroperoxyarachidonic acid. Highly purified prostaglandin H synthase, which processes both cyclo-oxygenase and hydroperoxidase activity, oxidized aminopyrine to the free radical. Purified prostaglandin H synthase reconstituted with Mn2+ protoporphyrin IX, which processes only cyclo-oxygenase activity, did not catalyze the formation of the aminopyrine free radical. Aminopyrine stimulated the reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid to 15-hydroxy-5,8,11-13-eicosatetraenoic acid. Approximately 1 molecule of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid was reduced for every 2 molecules of aminopyrine free radical formed, giving a stoichiometry of 1:2. The decay of the aminopyrine radical obeyed second-order kinetics. These results support the proposed mechanism in which aminopyrine is oxidized by prostaglandin H synthase hydroperoxidase to the aminopyrine free radical, which then disproportionates to the iminium cation. The iminium cation is further hydrolyzed to the demethylated amine and formaldehyde. Glutathione reduced the aminopyrine radical to aminopyrine with the concomitant oxidation of GSH to its thiyl radical as detected by ESR of the glutathione thiyl radical adduct. 相似文献
11.
The ESR spin trapping technique was used to study the first detectable radical intermediate in the oxidation of arachidonic acid by purified prostaglandin H synthase. The holoenzyme and the apoenzyme, reconstituted with either hematin or Mn2+ protoporphyrin IX, were investigated. Depending on the different types of enzyme activity present, arachidonic acid was oxidized to at least two free radicals. One of these radicals is thought to be the first ESR detectable radical intermediate in the conversion of arachidonic acid to prostaglandin G2 and was detected previously in incubations of ram seminal vesicle microsomes, which are rich in prostaglandin H synthase. The ESR findings correlated with oxygen incorporation into arachidonic acid and prostaglandin formation, where the spin trap inhibits oxygen incorporation and prostaglandin formation by apparently competing with oxygen for the carbon-centered radical. Substitution of arachidonic acid by octadeuterated (5, 6, 8, 9, 11, 12, 14, 15)-arachidonic acid confirmed that the radical adduct contained arachidonic acid that is bound to the spin trap at one of these eight positions. An attempt was made to explain the apparent time lag between the metabolic activity observed in the oxygraph measurements and the appearance of the trapped radical signals. 相似文献
12.
Methimazole, an irreversible, mechanism-based (suicide substrate) inhibitor of thyroid peroxidase and lactoperoxidase, also inhibits the oxidation of xenobiotics by prostaglandin hydroperoxidase. The mechanism(s) by which methimazole inhibits prostaglandin H synthase-catalyzed oxidations is not conclusively known. In studies reported here, methimazole inhibited the prostaglandin H synthase-catalyzed oxidation of benzidine, phenylbutazone, and aminopyrine in a concentration-dependent manner. Methimazole poorly supported the prostaglandin H synthase-catalyzed reduction of 5-phenyl-4-pentenyl hydroperoxide to the corresponding alcohol (5-phenyl-4-pentenyl alcohol), suggesting that methimazole is not serving as a competing reducing cosubstrate for the peroxidase. Methimazole is not a mechanism-based inhibitor of prostaglandin hydroperoxidase or horseradish peroxidase since methimazole did not inhibit the peroxidase-catalyzed, benzidine-supported reduction of 5-phenyl-4-pentenyl hydroperoxide. In contrast, methimazole inhibited the reduction of 5-phenyl-4-pentenyl hydroperoxide to 5-phenyl-4-pentenyl alcohol catalyzed by lactoperoxidase, confirming that methimazole is a mechanism-based inhibitor of that enzyme and that such inhibition can be detected by our assay. Glutathione reduces the aminopyrine cation free radical, the formation of which is catalyzed by the hydroperoxidase, back to the parent compound. Methimazole produced the same effect at concentrations equimolar to those required for glutathione. These data indicate that methimazole does not inhibit xenobiotic oxidations catalyzed by prostaglandin H synthase and horseradish peroxidase through direct interaction with the enzyme, but rather inhibits accumulation of oxidation products via reduction of a free radical-derived metabolite(s). 相似文献
13.
Nitric oxide and its derivatives have been shown to both activate and inhibit prostaglandin H(2) synthase 1 (PGHS-1). We set out to determine the mechanisms by which different nitrogen oxide derivatives modulate PGHS-1 activity. To this end, we show that 3-morpholinosydnonimine hydrochloride (SIN-1), a compound capable of generating peroxynitrite, activates purified PGHS-1 and also stimulates PGE(2) production in arterial smooth muscle cells in the presence of exogenous arachidonic acid. The effect of SIN-1 in smooth muscle cells was abrogated by superoxide and peroxynitrite inhibitors, which supports the hypothesis that peroxynitrite is an activating species of PGHS-1. Indeed, authentic peroxynitrite also induced PGE(2) production in arachidonic acid-stimulated cells. In contrast, when cells were exposed to the nitric oxide-releasing compound 1-hydroxy-2-oxo-3-[(methylamino)propyl]-3-methyl-1-triazene (NOC-7), PGHS-1 enzyme activity was inhibited in the presence of exogenous arachidonic acid. Finally, in lipid-loaded smooth muscle cells, we demonstrate that SIN-1 stimulates arachidonic acid-induced PGE(2) production; albeit, the extent of activation is reduced compared to that under normal conditions. These results indicate that formation of peroxynitrite is a key intermediary step in PGHS-1 activation. However, other forms of NO(x)() inhibit PGHS-1. These results may have implications in the regulation of vascular function and tone in normal and atherosclerotic arteries. 相似文献
14.
Marco Soriani Silvia Mazzuca Valentina Quaresima Maurizio Minetti 《Free radical biology & medicine》1993,14(6):589-599
Human neutrophils activatd by PMA were found to induced the formation of a nitroxide radical from DFO. The presence of SOD was necessary to permit the formation of the DFO radical. The inactive phorbol ester did not induce DFO radical, and
_sphinganine suppressed the radical produced by the active phorbol ester. Other cell stimuli (Zymocel and the chemotactic peptide) also induced the formation of the DFO radical, although radical concentration was very much lower than with PMA. Participation of .NO, ,OH or 1O2 was ruled out by the inability of NG-methyl-L-arginine, NG-nitro-L-arginine, DMSO, mannitol, histidine, and methionine to inhibit the formation of DFO radical produced by PMA-activated cells. Furthermore, PMA-activated cells dod not produce detectable levels of NO2−, as a stable oxidation product of .NO, and D2, which enhances the lifetime of singlet oxygen, did not modify the intensity or the lifetime of DFO radical. The involvement of cell MPO was suggested by the inhibition of the DFO radical observed after treatment with catalase or with antihuman MPO antibodies. Also, HOCI was found to induce the DFO radical in cell-free reactions, but our data indicate that the reaction leading to DFO radical formation by neutrophils involves the reduction of MPO compound II back to active enzyme (ferric-MPO). Anti-inflammatory drugs strongly increased the DFO radical produced by activated neutrophils. On the contrary, none of these drugs was able to increase the DFO radical produced by HOCl. Histidine and methionine that inhibited the DFO radical intensity in cell-free reactions, were shown to act directly onm HOCl. Experiments with MPO-H2O2 in SOD- and Cl−-free conditions showed the formation of DFO radical and confirmed the hypothesis of the involvement of compound II. The conversion of compound II to ferric MPO by DFO optimized the enzymatic activity of neurophils, and in the presence of monochlorodimedon (compound II promoting agent) we measured an increased HOCl production. When DFO was modified by conjugation with hydroxyethyl starch, it lost the ability to produce the radical either by neutrophils or by MPO-H2O2 and did not increase HOCl production. The inability of these DFO derivatives to produce potentially toxic species migh explain their reported lower toxicity in vivo. 相似文献
15.
Catherine Miller Lisa K Folkes Carolyn Mottley Peter Wardman Ronald P Mason 《Archives of biochemistry and biophysics》2002,397(1):113-118
There have been several conflicting reports as to the scavenging nature of glutathione toward the nitro radical anion of the drug nitrofurantoin. We produced the radical anion enzymatically using the xanthine oxidase/hypoxanthine system at pH 7.4 and pH 9.0 in the presence of various levels of glutathione from 10 to 100 mM and monitored any changes in the radical concentration via electron spin resonance spectroscopy. Independent of glutathione concentration, there was no decrease in the steady-state concentration of the radical. In fact, there was an average 30% increase in the concentration of the radical anion, which suggests enhanced enzyme activity in the presence of glutathione (GSH). These results, together with observations of the effects of glutathione on the stability of the radical anion generated by radiolysis or dithionite, rule out any detectable reaction between the nitrofurantoin radical anion and GSH under physiologically relevant conditions. 相似文献
16.
Isolation of the cDNA for human prostaglandin H synthase 总被引:5,自引:0,他引:5
Prostaglandin H Synthase (PGHS, cyclooxygenase) is a 67 kd protein which catalyzes the first step in prostaglandin synthesis. The primary amino acid sequence and the molecular mechanisms regulating expression are unknown. We report here isolation of a cDNA clone for the enzyme from human vascular endothelial cells for use in such studies. High titre, polyclonal antiserum against PGHS was developed in rabbits. The antiserum was monospecific, reacted with cyclooxygenase on Western blots at a limiting dilution of 1:500,000 and immunoprecipitated cyclooxygenase synthesized by in vitro translation of PGHS messenger RNA. It was used to screen a lambda gt11 cDNA expression library from human endothelial cells. Three positive clones were isolated. Following plaque purification, one clone reacted strongly with two other polyclonal antisera independently raised against highly purified cyclooxygenase and the aspirin-acetylated enzyme. Western blot analysis confirmed production of a large approximately 180 kd fusion protein of cyclooxygenase and beta-galactosidase. The cDNA insert of approximately 2.2 kilo base pairs was excised and subcloned into plasmid pUC8. A 24 nucleotide DNA probe, synthesized according to the amino acid sequence of the aspirin-acetylation site of cyclooxygenase, hybridized strongly with the 2.2 kbp cDNA insert. It is concluded that the 2.2 kbp cDNA insert represents a cDNA clone for human cyclooxygenase, which also expresses the aspirin-acetylation site. This is the first reported isolation of the cDNA for this enzyme, and will facilitate further studies on the primary sequence and on the regulation of the enzyme at the molecular level. 相似文献
17.
Quin2 and its analogs BAPTA, 5,5'-dimethyl BAPTA, 5,5'-difluoro BAPTA, fura-2, and indo-1 were developed to measure intracellular calcium concentrations. In this study we investigated whether quin2 and its analogs are susceptible to peroxidase-catalyzed oxidation. The hydroperoxidase activity of prostaglandin H synthase, like other peroxidases, is capable of oxidizing a wide variety of substrates. It was found that quin2 and its analogs served as reducing cofactors for the hydroperoxidase activity of prostaglandin H synthase, undergoing oxidation in the process. Furthermore, arachidonic acid metabolism was stimulated. Oxidation of quin2 and its analogs resulted in the formation of a carbon-centered radical, as could be detected by ESR, and in the formation of formaldehyde. Quin2 fluorescence decreased upon addition of arachidonic acid and prostaglandin H synthase. Furthermore, addition of calcium no longer resulted in an increase in quin2 fluorescence, as was observed prior to the addition of arachidonic acid and the enzyme. This indicates that one or more of the -N-CH2-COOH groups, which are responsible for the binding of calcium, were oxidized by the hydroperoxidase. Since prostaglandin H synthase is present in many cellular systems in which calcium concentrations are modulated, oxidation of the calcium probe might not only affect the measurement of intracellular calcium but could activate arachidonic acid metabolism as well. 相似文献
18.
19.
T E Eling W C Glasgow J F Curtis W C Hubbard J A Handler 《The Journal of biological chemistry》1991,266(19):12348-12355
Prostaglandin H synthase oxidizes arachidonic acid to prostaglandin G2 (PGG2) via its cyclooxygenase activity and reduces PGG2 to prostaglandin H2 by its peroxidase activity. The purpose of this study was to determine if endogenously generated PGG2 is the preferred substrate for the peroxidase compared with exogenous PGG2. Arachidonic acid and varying concentrations of exogenous PGG2 were incubated with ram seminal vesicle microsomes or purified prostaglandin H synthase in the presence of the reducing cosubstrate, aminopyrine. The formation of the aminopyrine cation free radical (AP.+) served as an index of peroxide reduction. The simultaneous addition of PGG2 with arachidonic acid did not alter cyclooxygenase activity of ram seminal vesicle microsomes or the formation of the AP.+. This suggests that the formation of AP.+, catalyzed by the peroxidase, was supported by endogenous endoperoxide formed from arachidonic acid oxidation rather than by the reduction of exogenous PGG2. In addition to the AP.+ assay, the reduction of exogenous versus endogenous PGG2 was studied by using [5,6,8,9,11,12,14,15-2H]arachidonic acid and unlabeled PGG2 as substrates, with gas chromatography-mass spectrometry techniques to measure the amount of reduction of endogenous versus exogenous PGG2. Two distinct results were observed. With ram seminal vesicle microsomes, little reduction of exogenous PGG2 was observed even under conditions in which all of the endogenous PGG2 was reduced. In contrast, studies with purified prostaglandin H synthase showed complete reduction of both exogenous and endogenous PGG2 using similar experimental conditions. Our findings indicate that PGG2 formed by the oxidation of arachidonic acid by prostaglandin H synthase in microsomal membranes is reduced preferentially by prostaglandin H synthase. 相似文献
20.
Requirements for hydroperoxide by the cyclooxygenase and peroxidase activities of prostaglandin H synthase 总被引:2,自引:0,他引:2
Purified prostaglandin H synthase contains cyclooxygenase activity that forms the hydroperoxide, prostaglandin G, and peroxidase activity which removes hydroperoxides. Since hydroperoxides are necessary activators of cyclooxygenase activity, the paradoxical presence of two apparently opposing activities requires careful interpretation. Kinetic studies indicate that the concentration of hydroperoxide needed for full cyclooxygenase activity is much less than that which gives 50 percent effectiveness with the peroxidase. Thus, the peroxidase activity of the synthase is very ineffective in decreasing the hydroperoxide concentration below levels that still permit rapid cyclooxygenase action. 相似文献