Prostaglandin G2 levels during reaction of prostaglandin H synthase with arachidonic acid

Prostaglandin H synthase catalyzes the formation of prostaglandin (PG) G2 from arachidonic acid (cyclooxygenase activity), and also the reduction of PGG2 to PGH2 (peroxidase activity). The ability of the pure synthase to accumulate the hydroperoxide, PGG2, under conditions allowing the concurrent function of both catalytic activities was investigated. The peroxidase velocity was continuously determined from the absorbance increases at 611 nm that accompanied oxidation of a peroxidase cosubstrate, N,N,N',N'-tetramethylphenylenediamine, and PGG2 concentrations were calculated from the peroxidase velocities and the peroxidase Vmax and Km values. Cyclooxygenase velocities were then calculated from the changes in PGG2. Parallel reactions monitored by the use of radiolabelled arachidonate or with a polarographic oxygen electrode were used to confirm the calculated PGG2 levels and the cyclooxygenase velocities. The concentration of PGG2 was found to follow a transient course as the reaction of the synthase progressed, rapidly rising to a maximum of 0.7 microM in the first 10 s, and then declining slowly, reaching 0.1 microM after 60 s. The maximal level of PGG2 achieved during the reaction was constant at about 0.7 microM with higher amounts of added cyclooxygenase capacity (0.3-0.6 microM PGG2/s) but was only about 0.4 microM when the added cyclooxygenase capacity was 0.1 microM PGG2/s. The peroxidase was found to lose only 30% of its activity after 90 s, a point where the cyclooxygenase was almost completely inactive. These results support the concept of a burst of catalytic action from the cyclooxygenase and a reactive, more sustained, catalytic action from the peroxidase during the reaction of the synthase with arachidonic acid.     

Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid

Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O(2), and two electrons to prostaglandin endoperoxide H(2) (PGH(2)). Formation of PGH(2) involves an initial oxygenation of arachidonate to yield PGG(2) catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG(2) to form PGH(2) catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co(3+)-heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG(2) versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually make less but measurable contributions to optimal catalytic efficiency.     

Studies on the reduction of endogenously generated prostaglandin G2 by prostaglandin H synthase

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.     

Prostaglandin endoperoxide H synthases: peroxidase hydroperoxide specificity and cyclooxygenase activation

The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.     

Radical scavenging as the mechanism for stimulation of prostaglandin cyclooxygenase and depression of inflammation by lipoic acid and sodium iodide.

Certain radical-trapping reducing agents have been shown to stimulate prostaglandin biosynthesis in vitro (1--6) and to depress phorbol myristate acetate-induced mouse ear edema (16). The increased prostaglandin synthesis resulted from influences on the cyclooxygenase. To ascertain whether these alterations were due to direct interaction with the enzyme or to indirect scavenging of the oxidant released during PGG2 reduction, we report the effects of lipoic acid and sodium iodide. Both of these agents stimulated the enzymatic oxygenation of arachidonic acid, increased the reduction of PGG2 to PGH2, quenched the EPR signal induced by arachidonic acid and depressed mouse ear edema. In addition to discovering two unusual antiinflammatory agents, we have confirmed that materials with entirely different structures can have identical effects on the cyclooxygenase, suggesting indirect stimulation of this enzyme due to trapping of the oxidant.     

Different suicide inactivation processes for the peroxidase and cyclooxygenase activities of prostaglandin endoperoxide H synthase-1.

Prostaglandin endoperoxide H synthases (PGHSs)-1 and -2 have a cyclooxygenase (COX) activity involved in forming prostaglandin G2 (PGG2) from arachidonic acid and an associated peroxidase (POX) activity that reduces PGG2 to PGH2. Suicide inactivation processes are observed for both POX and COX reactions. Here we report COX reaction conditions for PGHS-1 under which complete COX inactivation occurs but with > or = 60% retention of POX activity. The rates of POX inactivation were compared for native oPGHS-1 versus Y385F oPGHS-1, a mutant that cannot form the Tyr385 radical of COX Intermediate II; the rates were the same for both native and Y385F oPGHS-1. Our data indicate that a COX Intermediate II/acyl or product complex is the precursor in COX inactivation. However, another species, probably an Intermediate II-like species but with a radical centered on a tyrosine other than Tyr385, is the immediate precursor for POX inactivation.     

Involvement of phospholipid hydroperoxide glutathione peroxidase in the modulation of prostaglandin D2 synthesis

Antigenic cross-linking of the high affinity IgE receptors on mast cells induced the synthesis of prostaglandin D(2) (PGD(2)). The production of PGD(2) in L9 cells, which overexpressed non-mitochondrial phospholipid glutathione peroxidase (PHGPx), was only one-third that in the control line of cells (S1 cells). The reduction in the formation of PGD(2) in L9 cells was reversed upon inhibition of PHGPx activity by buthionine sulfoximine. Experiments with inhibitors demonstrated that prostaglandin H synthase-2 (PGHS-2) was the isozyme responsible for the production of PGD(2) upon cross-linking of IgE receptors. The conversion of radiolabeled arachidonic acid to prostaglandin H(2) (PGH(2)) was strongly inhibited in L9 cells, whereas the rate of conversion of PGH(2) to PGD(2) was the same in L9 cells and S1 cells, indicating that PGHS was inactivated in L9 cells. The PGHS activity in L9 cells was about half that in S1 cells. However, PGHS activity in L9 cells increased to the level in S1 cells upon the addition of the hydroperoxide 15-hydroperoxyeicosatetraenoic acid or of 3-chloroperoxybenzoic acid. These results suggest that non-mitochondrial PHGPx might be involved in the inactivation of PGHS-2 in nucleus and endoplasmic reticulum via reductions in levels of the hydroperoxides that are required for full activation of PGHS. Therefore, it appears that PHGPx might function as a modulator of the production of prostanoids, in addition to its role as an antioxidant enzyme.     

Quantitative studies of hydroperoxide reduction by prostaglandin H synthase. Reducing substrate specificity and the relationship of peroxidase to cyclooxygenase activities

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)     

Prostaglandin H synthase: distinct binding sites for cyclooxygenase and peroxidase substrates

Prostaglandin H synthase has two distinct catalytic activities: a cyclooxygenase activity that forms prostaglandin G2 from arachidonic acid; and a peroxidase activity that reduces prostaglandin G2 to prostaglandin H2. Lipid hydroperoxides, such as prostaglandin G2, also initiate the cyclooxygenase reaction, probably via peroxidase reaction cycle enzyme intermediates. The relation between the binding sites for lipid substrates of the two activities was investigated with an analysis of the effects of arachidonic and docosahexaenoic acids on the reaction kinetics of the peroxidase activity, and their effects on the ability of a lipid hydroperoxide to initiate the cyclooxygenase reaction. The cyclooxygenase activity of pure ovine synthase was found to have an apparent Km value for arachidonate of 5.3 microM and a Ki value (competetive inhibitor) for docosahexaenoate of 5.2 microM. When present at 20 microM neither fatty acid had a significant effect on the apparent Km value of the peroxidase for 15-hydroperoxyeicosatetraenoic acid: the values were 7.6 microM in the absence of docosahexaenoic acid and 5.9 microM in its presence, and (using aspirin-treated synthase) 13.7 microM in the absence of arachidonic acid and 15.7 microM in its presence. Over a range of 1 to 110 microM the level of arachidonate had no significant effect on the initiation of the cyclooxygenase reaction by 15-hydroperoxyeicosatetraenoic acid. The inability of either arachidonic acid or docosahexaenoic acid to interfere with the interaction between the peroxidase and lipid hydroperoxides indicates that the cyclooxygenase and peroxidase activities of prostaglandin H synthase have distinct binding sites for their lipid substrates.     

The cyclooxygenase hydroperoxide product PGG(2) activates synaptic nitric oxide synthase: a possible antioxidant response to membrane lipid peroxidation

Nitric oxide is a potent inhibitor of membrane lipid peroxidation. It is unknown, however, whether nitric oxide synthase (NOS) activity increases under conditions of membrane lipid peroxidation. Importantly, cyclooxygenase (COX)-catalyzed peroxidation of arachidonic acid is well-established to be increased by lipid hydroperoxides. The results of the present study demonstrate that the COX hydroperoxide product prostaglandin G(2) (PGG(2)) greatly stimulated NOS activity in synaptosomal membrane fractions from rat brain in a dose-dependent (EC(50) = 0.2 microM) manner in the presence of ATP and the antioxidant urate. NOS activation was also produced, albeit to a lesser extent, by 15-hydroperoxyeicosatetraenoic acid (15-HPETE) but not by the corresponding hydroxy compounds PGH(2) and 15-HETE or by hydrogen peroxide. These findings demonstrate that PGG(2)-activated synaptic NOS by a hydroperoxide-mediated pathway and support the view that NOS activation may be an important physiological response to lipid peroxidation.     

Enzymological and immunological studies on a clinical case of platelet cyclooxygenase abnormality

A peroxidase-linked immunoassay of the sandwich type was developed for a quantitative determination of the amount of human cyclooxygenase. Two species of monoclonal antibodies (hPES01 against the human enzyme and PES-5 against the bovine enzyme) were utilized, which recognized different epitopes on the cyclooxygenase of human platelets. The peroxidase activity of the immunoprecipitate was correlated with the amount of cyclooxygenase. The enzyme immunoassay was applied to platelets from 15 normal subjects and a clinical case of platelet cyclooxygenase abnormality with a prolonged bleeding time. Almost the same level of immunoreactive protein was found in platelets of both normal subjects and the patient. However, the solubilized enzyme from the patient's platelets did not transform arachidonic acid to prostaglandin H2 (PGH2) while thromboxane production from PGH2 was observed at a normal level.     

Histidine 386 and its role in cyclooxygenase and peroxidase catalysis by prostaglandin-endoperoxide H synthases

Prostaglandin-endoperoxide H synthases (PGHSs) have a cyclooxygenase that forms prostaglandin (PG) G2 from arachidonic acid (AA) plus oxygen and a peroxidase that reduces the PGG2 to PGH2. The peroxidase activates the cyclooxygenase. This involves an initial oxidation of the peroxidase heme group by hydroperoxide, followed by oxidation of Tyr385 to a tyrosyl radical within the cyclooxygenase site. His386 of PGHS-1 is not formally part of either active site, but lies in an extended helix between Tyr385, which protrudes into the cyclooxygenase site, and His388, the proximal ligand of the peroxidase heme. When His386 was substituted with alanine in PGHS-1, the mutant retained <2.5% of the native peroxidase activity, but >20% of the native cyclooxygenase activity. However, peroxidase activity could be restored (10-30%) by treating H386A PGHS-1 with cyclooxygenase inhibitors or AA, but not with linoleic acid; in contrast, mere occupancy of the cyclooxygenase site of native PGHS-1 had no effect on peroxidase activity. Heme titrations indicated that H386A PGHS-1 binds heme less tightly than does native PGHS-1. The low peroxidase activity and decreased affinity for heme of H386A PGHS-1 imply that His386 helps optimize heme binding. Molecular dynamic simulations suggest that this is accomplished in part by a hydrogen bond between the heme D-ring propionate and the N-delta of Asn382 of the extended helix. The structure of the extended helix is, in turn, strongly supported by stable hydrogen bonding between the N-delta of His386 and the backbone carbonyl oxygens of Asn382 and Gln383. We speculate that the binding of cyclooxygenase inhibitors or AA to the cyclooxygenase site of ovine H386A PGHS-1 reopens the constriction in the cyclooxygenase site between the extended helix and a helix containing Gly526 and Ser530 and restores native-like structure to the extended helix. Being less bulky than AA, linoleic acid is apparently unable to reopen this constriction.     

Isolation and properties of enzymes involved in prostaglandin biosynthesis.

Prostaglandin (PG) endoperoxide synthetase was purified until homogeneity had been attained. The pure enzyme displays both cyclooxygenase and peroxidase activity, in accordance with the work of MIYAMOTO et al. (J. biol. Chem. 252, 2629--2636 (1976)). This enzyme therefore converts arachidonic acid into PGH2. Glutathione S-transferases, in the presence of glutathione, convert PGH2 into a mixture of PGF2alpha, PGE2 and PGD2. A new transferase in sheep lung gives mainly PGF2alpha and PGD2. Isolation and properties of these enzymes will be discussed. Finally, progress will be reported on the isolation of a soluble enzyme from various rat organs such as lung and spleen, which forms almost exclusively prostaglandin D.     

Substitution of tyrosine for the proximal histidine ligand to the heme of prostaglandin endoperoxide synthase 2: implications for the mechanism of cyclooxygenase activation and catalysis

Prostaglandin H(2) synthesis by prostaglandin endoperoxide synthase (PGHS) requires the heme-dependent activation of the protein's cyclooxygenase activity. The PGHS heme participates in cyclooxygenase activation by accepting an electron from Tyr385 located in the cyclooxygenase active site. Two mechanisms have been proposed for the oxidation of Tyr385 by the heme iron: (1) ferric enzyme oxidizes a hydroperoxide activator and the incipient peroxyl radical oxidizes Tyr385, or (2) ferric enzyme reduces a hydroperoxide activator and the incipient ferryl-oxo heme oxidizes Tyr385. The participation of ferrous PGHS in cyclooxygenase activation was evaluated by determining the reduction potential of PGHS-2. Under all conditions tested, this potential (<-135 mV) was well below that required for reactions leading to cyclooxygenase activation. Substitution of the proximal heme ligand, His388, with tyrosine was used as a mechanistic probe of cyclooxygenase activation. His388Tyr PGHS-2, expressed in insect cells and purified to homogeneity, retained cyclooxygenase activity but its peroxidase activity was diminished more than 300-fold. Concordant with this poor peroxidase activity, an extensive lag in His388Tyr cyclooxygenase activity was observed. Addition of hydroperoxides resulted in a concentration-dependent decrease in lag time consistent with each peroxide's ability to act as a His388Tyr peroxidase substrate. However, hydroperoxide treatment had no effect on the maximal rate of arachidonate oxygenation. These data imply that the ferryl-oxo intermediates of peroxidase catalysis, but not the Fe(III)/Fe(II) couple of PGHS, are essential for cyclooxygenase activation. In addition, our findings are strongly supportive of a branched-chain mechanism of cyclooxygenase catalysis in which one activation event leads to many cyclooxygenase turnovers.     

Levuglandinyl adducts of proteins are formed via a prostaglandin H2 synthase-dependent pathway after platelet activation

The product of oxygenation of arachidonic acid by the prostaglandin H synthases (PGHS), prostaglandin H(2) (PGH(2)), undergoes rearrangement to the highly reactive gamma-ketoaldehydes, levuglandin (LG) E(2), and LGD(2). We have demonstrated previously that LGE(2) reacts with the epsilon-amine of lysine to form both the levuglandinyl-lysine Schiff base and the pyrrole-derived levuglandinyl-lysine lactam adducts. We also have reported that these levuglandinyl-lysine adducts are formed on purified PGHSs following the oxygenation of arachidonic acid. We now present evidence that the levuglandinyl-lysine lactam adduct is formed in human platelets upon activation with exogenous arachidonic acid or thrombin. After proteolytic digestion of the platelet proteins, and isolation of the adducted amino acid residues, this adduct was identified by liquid chromatography-tandem mass spectrometry. We also demonstrate that formation of these adducts is inhibited by indomethacin, a PGHS inhibitor, and is enhanced by an inhibitor of thromboxane synthase. These data establish that levuglandinyl-lysine adducts are formed via a PGHS-dependent pathway in whole cells, even in the presence of an enzyme that metabolizes PGH(2). They also demonstrate that a physiological stimulus is sufficient to lead to the lipid modification of proteins through the levuglandin pathway in human platelets.     

Aromatic hydroxamic acids and hydrazides as inhibitors of the peroxidase activity of prostaglandin H2 synthase-2

The cyclooxygenase activity of the bifunctional enzyme prostaglandin H(2) synthase-2 (PGHS-2) is the target of non-steroidal anti-inflammatory drugs. Inhibition of the peroxidase activity of PGHS has been less studied. Using Soret absorption changes, the binding of aromatic hydroxamic acids to the peroxidase site of PGHS-2 was examined to investigate the structural determinants of inhibition. Typical of mammalian peroxidases, the K(d) for benzhydroxamic acid (42mM) is much greater than that for salicylhydroxamic acid (475microM). Binding of the hydroxamic acid tepoxalin (25microM) resulted in only minor Soret changes. However, tepoxalin is an efficient reducing cosubstrate, indicating that it is an alternative electron donor rather than an inhibitor of the peroxidase activity. Aromatic hydrazides are metabolically activated inhibitors of peroxidases. 2-Naphthoichydrazide (2-NZH) caused the time- and concentration-dependent inhibition of both PGHS-2 peroxidase and cyclooxygenase activities. H(2)O(2) was required for the inactivation of both PGHS-2 activities and indomethacin (which binds at the cyclooxygenase site) did not affect the peroxidase inhibitory potency of 2-NZH. A series of aromatic hydrazides were found to be potent inhibitors of PGHS-2 peroxidase activity with IC(50) values in the 6-100microM range for 13 of the 18 hydrazides examined. Selective inhibition of PGHS-2 over myeloperoxidase and horseradish peroxidase isozyme C was increased by certain ring substitutions. In particular, a chloro group para to the hydrazide moiety increased the PGHS-2 selectivity relative to both myeloperoxidase and horseradish peroxidase isozyme C.     

Prostaglandin synthases: recent developments and a novel hypothesis

Cells are continuously exposed to cues, which signal cell survival or death. Fine-tuning of these conflicting signals is essential for tissue development and homeostasis, and defective pathways are linked to many disease processes, especially cancer. It is well established that prostaglandins (PGs), as signalling molecules, are important regulators of cell proliferation, differentiation and apoptosis. PG production has been a focus of many researchers interested in the mechanisms of parturition. Previously, investigators have focussed on the committed step of PG biosynthesis, the conversion by prostaglandin H synthase (PGHS; also termed cyclo-oxygenase, COX) of arachidonic acid (AA) (substrate) to PGH2, the common precursor for biosynthesis of the various prostanoids. However, recently the genes encoding the terminal synthase enzymes involved in converting PGH2 to each of the bioactive PGs, including the major uterotonic PGs, PGE2 (PGE synthase) and PGF2alpha (PGF synthase), have been cloned and characterized. This review highlights how the regulation of the expression and balance of key enzymes can produce, from a single precursor, prostanoids with varied and often opposing effects.     

Acetaminophen and analogs as cosubstrates and inhibitors of prostaglandin H synthase

Previous studies have shown that acetaminophen (APAP) is converted by prostaglandin H synthase (PGHS) to both one-electron oxidized products and the two-electron oxidized product, N-acetyl-p-benzoquinone imine (NAPQI). The present study further characterizes this reaction and shows that relatively low concentrations (20-200 microM) of APAP stimulate PGHS activity in ram seminal vesicle microsomes, whereas high concentrations (greater than 10 mM) inhibit the conversion of arachidonic acid (AA) to 15-hydroperoxy-9,11-peroxidoprosta-5,13-dienoic acid (PGG2). Stimulatory and inhibitory activities apparently involve the reduction of oxidized complexes of PGHS, and stimulatory and inhibitory activities roughly correlate with the electrochemical half-wave oxidation potentials of a series of hydroxyacetanilides. Using APAP as a probe, it was found that at low concentrations, APAP is converted in a cooxidation reaction with arachidonic acid to a dimer, 4'4"'-dihydroxy-3', 3"'-biacetanilide (bi-APAP), and other polymeric products. Moreover, an electrophilic metabolite of acetaminophen, NAPQI, was detected directly and also detected indirectly by its reaction with glutathione (GSH) to form 3'-(S-glutathionyl)acetaminophen (GS-APAP). The formation of all products was inhibited by indomethacin and the reductants, ascorbic acid and butylated hydroxyanisole (BHA). However, in the presence of GSH, ascorbic acid only partially inhibited the formation of GS-APAP while almost completely inhibiting the formation of bi-APAP. The same products of APAP (bi-APAP and NAPQI) were formed by PGHS and hydrogen peroxide in reactions that were not inhibited by indomethacin. At high concentrations of APAP that inhibit PGHS, the formation of products in the presence of arachidonic acid but not H2O2 was inhibited. These findings are generally consistent with a mechanism of acetaminophen oxidation by PGHS that involves common intermediate enzyme forms for both cyclooxygenase- and hydroperoxidase-catalyzed reactions. At least one of the intermediate complexes is reduced by relatively low concentrations of APAP and stimulates PGHS, whereas another intermediate complex is reduced by APAP at higher concentrations to inhibit the enzyme.     

Cyclooxygenase and peroxidase inactivation of prostaglandin-H-synthase during catalysis

Prostaglandin-H-synthase (PGHS) is a bifunctional enzyme catalyzing cyclooxygenase and peroxidase reactions and undergoing irreversible inactivation during catalysis. A new method for kinetic studies of both PGHS activities in the course of cyclooxygenase as well as peroxidase reactions and also preincubation with hydroperoxides is suggested. It is shown that peroxidase activity is retained after complete cyclooxygenase inactivation and cyclooxygenase activity is retained after complete peroxidase inactivation. Two-stage cyclooxygenase inactivation occurs on preincubation of PGHS with hydrogen peroxide. Studies on inactivation under various conditions indicate that chemical mechanisms of cyclooxygenase and peroxidase inactivation are different. The data allow development of kinetic models.     

Antithrombotic and antineoplastic effects of phyto-organosulfur compounds

The antiplatelet activity of methyl allyltrisulfide (MATS), a component commonly present in steam-distilled garlic oil, has been demonstrated by the authors. MATS inhibits arachidonic acid cascade at the reaction site with PGH synthase. However, this enzyme catalyzes two successive reactions, from arachidonic acid to PGG2, and from PGG2 to PGH2. The present study revealed that MATS inhibited the latter reaction. In addition, our recent findings that to a promyelocytic leukemia cell HL60, Allium oils shows marked anti-neoplastic effects representing both growth suppression and differentiation activities are described. The garlic oil and onion oil showed almost equal ability in inducing the proliferation, which measured either by nitroblue-tetrazolium reducing activity assay or by flow cytometry for detecting CD11b expression. The combination use of one of these oils with all-trans retinoic acid or with dimethyl sulfoxide lead to the marked differentiation of the cells, and their effects were estimated to be synergistic.