Metabolism of 5(6)Oxidoeicosatrienoic acid by ram seminal vesicles. Formation of two stereoisomers of 5-hydroxyprostaglandin I1

Cytochrome P-450 can metabolize arachidonic (5,8,11,14-eicosatetraenoic) acid to four epoxides. One of them, cis-5(6)oxido-8,11,14-eicosatrienoic acid, has been reported to possess biological activity. To ascertain whether this epoxide could be a substrate for the enzyme fatty acid cyclooxygenase, synthetic 3H-labeled cis-5(6)-oxido-8,11,14-eicosatrienoic acid was incubated with microsomes of ram seminal vesicles and incubated with microsomes of ram seminal vesicles and the products were separated by reversed phase high performance liquid chromatography. The substrate was enzymatically transformed into products, which were more polar than 5,6-dihydroxy-8,11,14-eicosatrienoic acid. The biosynthesis was strongly inhibited by indomethacin or diclofenac sodium, two inhibitors of fatty acid cyclooxygenase. Two of the major metabolites could be identified by capillary gas chromatography-mass spectrometry as two stereoisomers of 5-hydroxyprostaglandin I1, viz. (5R,6R)-5-hydroxyprostaglandin I1 and (5S,6S)-5-hydroxyprostaglandin I1. The structures were established by comparison with the mass spectra of authentic material and by the retention time on capillary gas chromatography using deuterated internal standards. The two stereoisomers were presumably formed nonenzymatically from the intermediate 5(6)oxidoprostaglandin endoperoxides or from 5(6)oxidoprostaglandin F1 alpha during the isolation procedure.     

Isolation and biosynthesis of 20-hydroxyprostaglandins E1 and E2 in ram seminal fluid

Ram semen was found to contain 20-hydroxyprostaglandin E1 and 20-hydroxyprostaglandin E2. The relative amounts of the two compounds were almost equal, although ram semen contained at least 10 times more prostaglandin E1 than prostaglandin E2. The accessory genital glands of the ram were analyzed for their capacity to metabolize [14C]arachidonic acid to prostaglandins. Biosynthesis of prostaglandins was only found in microsomes of the mucosa of the ampulla of vas deferens and in microsomes of the vesicular glands. Ram vesicular glands and the ampulla of vas deferens were also found to contain the two 20-hydroxylated E prostaglandins. Microsomes of ram vesicular glands and NADPH metabolized exogenous prostaglandin E2 to 20-hydroxyprostaglandin E2 albeit in low yields. Prostaglandin E2 appeared to be a better substrate than prostaglandin E1. Microsomes of human seminal vesicles and NADPH metabolized exogenous prostaglandin E2 to 19-hydroxyprostaglandin E2. The results show that 19- and 20-hydroxylation of prostaglandins occurs in human and ram seminal vesicles, respectively, and possibly also in the ampulla of vas deferens of the ram. The ram and human enzymes specifically hydroxylated the terminal and the penultimate carbon of prostaglandin E2, respectively.     

Biochemical characterization of prostaglandin 19-hydroxylase of seminal vesicles

The microsomal fraction of homogenates of seminal vesicles of men and monkeys, Macaca fascicularis, were analyzed for prostaglandin (PG) 19-hydroxylase activity. The microsomes of the monkey seminal vesicles, supplemented with 1 mM NADPH, metabolized 0.2 mM PGE1 to 19-hydroxy-PGE1 at a mean rate of 0.26 nmol/min/mg of protein (with an apparent Km and an apparent Vmax of 40 microM and 0.30 nmol/min/mg of protein, respectively). The enzyme catalyzed the incorporation of atmospheric oxygen into the substrate. Substituting NADH for NADPH reduced the prostaglandin E1 19-hydroxylase activity to 40%. Carbon monoxide and proadifen (SKF 525A) inhibited the enzyme. Prostaglandin E2 (0.2 mM) was metabolized to 19-hydroxyprostaglandin E2 (0.2 nmol/min/mg of protein), but PGE1 was preferred as a substrate. Prostaglandin B1 was metabolized to 18-hydroxy-, 19-hydroxy-, and 20-hydroxyprostaglandin B1 at a combined rate of approximately 25% of prostaglandin E1. 19-Hydroxyprostaglandin B1 was the main product. The microsomes of human seminal vesicles metabolized 0.2 mM PGE2 to 19-hydroxy-PGE2 in the presence of 1 mM NADPH, while carbon monoxide inhibited this reaction. These results suggest that prostaglandin 19-hydroxylase of seminal vesicles might be a cytochrome P-450. The biosynthesis of 19-hydroxyprostaglandin E1 and 19-hydroxyprostaglandin E2 was also studied in vivo in man by analysis of the product/substrate ratios (i.e. 19-hydroxyprostaglandin E1/prostaglandin E1 and 19-hydroxyprostaglandin E2/prostaglandin E2) in a series of consecutive ejaculates, which were obtained during short intervals. There was a 10-fold interindividual difference in these ratios. Although the product/substrate ratios decreased, the 19-hydroxylation of E prostaglandins appeared to be efficient in vivo, which was in contrast to the rather slow biosynthesis in vitro.     

Biosynthesis of 18(RD)-hydroxyeicosatetraenoic acid from arachidonic acid by microsomes of monkey seminal vesicles. Some properties of a novel fatty acid omega 3-hydroxylase and omega 3-epoxygenase

Microsomes of seminal vesicles of the cynomolgus monkey were incubated with [14C]5,8,11,14-eicosatetraenoic (arachidonic) acid and NADPH for 40 min at 37 degrees C and the products were characterized. Prostaglandins F2 alpha and E2 were the two main metabolites (approximately 52% of radioactivity), while 18(R)-hydroxy-cis-5,8,11,14-eicosatetraenoic acid (18(R)-HETE) was identified as the main, less polar product (approximately 13%). Significant biosynthesis of the 19-hydroxy or 20-hydroxy metabolites of arachidonic acid could not be detected. The formation of 18(R)-HETE was further investigated in the presence of a prostaglandin synthesis inhibitor, diclofenac sodium. The omega 3-hydroxylation was only partly supported by substituting NADH for NADPH. The hydroxyl oxygen of 18(R)-HETE was derived from the atmosphere and the omega 3-hydroxylation was inhibited by proadifen and partly inhibited by carbon monoxide. These findings suggest that 18(R)-HETE is formed by a cytochrome P-450 (P-450 omega 3). Linoleic acid and 8,11,14-eicosatetraenoic acid were also substrates of the enzyme, but stearic acid was not metabolized. 5,8,11,14,17-Eicosatetraenoic acid was oxygenated under these conditions mainly to 17,18-dihydroxy-5,8,11,14-eicosatetraenoic acid, presumably formed from 17(18)-epoxy-5,8,11,14-eicosatetraenoic acid by hydrolysis. The seminal microsomes thus seem to possess both omega 3-hydroxylase and omega 3-epoxygenase activity. These seminal vesicles also contain prostaglandin E 19-hydroxylase (Oliw, E.H., Kinn, A.-C., and Kvist, U. (1988) J. Biol. Chem. 263, 7222-7227). The presence of arachidonate omega 3-hydroxylase and prostaglandin E 19-hydroxylase was assessed in microsomes of adult and juvenile monkey livers. Arachidonic acid was metabolized extensively to diols (via epoxides), but 18-HETE could not be detected. In contrast, prostaglandin E1 was slowly hydroxylated mainly to 19-hydroxyprostaglandin E1 by both adult male and female juvenile hepatic microsomes. The results indicate that P-450 omega 3 of seminal vesicles might be a tissue-specific enzyme.     

Identification and biological activity of a novel natural prostaglandin, 5,6-dihydro-prostaglandin E3

A novel natural E-prostaglandin was detected by HPLC among the endogenous prostaglandins extracted from ram seminal vesicles. The corresponding precursor - all-cis-eicosa-8, 11, 14, 17-tetraenoic acid was isolated from bovine liver lipids and the preparative biosynthesis with the microsomal fraction of ram seminal vesicles was performed. The isolated product was purified by HPLC and identified by GC-MS as 5,6-dihydro-PGE3. The results of in vitro tests demonstrate that 5,6-dihydro-PGE3 is 14 times less active uterine stimulant than PGE1, at the same time retaining 75% of the anti-aggregatory potency of PGE1. Thus, 5,6-dihydro-PGE3 meets the requirements of a selective antithrombotic agent more than PGE1.     

Metabolism of polyunsaturated (n-3) fatty acids by monkey seminal vesicles: isolation and biosynthesis of omega-3 epoxides.

Monooxygenases of monkey seminal vesicles can metabolize arachidonic acid (20:4(n-6)) by w3-hydroxylation to 18(R)-hydroxyeicosatetraenoic acid (18(R)-HETE) and eicosapentaenoic acid (20:5(n-3)) to 17,18-dihydroxyeicosatetraenoic acid (Oliw, E.H. (1989) J. Biol. Chem. 264, 17845-17853). The present study aimed to further characterize the oxygenation of (n-3) polyunsaturated fatty acids. 14C-Labelled 22:6(n-3), 20:5(n-3), 20:4-(n-3) and 18:3(n-3) were incubated with microsomes of seminal vesicles of the cynomolgus monkey, NADPH and a cyclooxygenase inhibitor, diclofenac, and the main metabolites were identified by capillary gas chromatography-mass spectrometry. 22:6(n-3) was slowly metabolized to 19,20-dihydroxy-4,7,10,13,16-docosapentaenoic acid, while 20:5(n-3), 20:4(n-3) and 18:3(n-3) were metabolized more efficiently to the corresponding w4,w3-diols. The w3 epoxides, which were obtained from 20:5(n-3) and 18:3(n-3), were isolated in the presence of an epoxide hydrolase inhibitor, 1(2)epoxy-3,3,3-trichloropropane, and the geometry of the epoxides was determined to be 17S, 18R and 15S, 16R, respectively. While 20:5(n-3) was metabolized almost exclusively to the epoxide and diol pair of metabolites, 18:3(n-3) was metabolized not only to the w3 epoxide and the corresponding diol, but also to the w2 alcohol, 17(R)-hydroxy-9,12,15-octadecatrienoic acid. 22:6(n-3) and 5,8,11,14-eicosatetraynoic acid inhibited the biosynthesis of 18(R)-HETE from arachidonic acid (IC50 0.16 and 0.14 mM, respectively). In comparison with 20:4 or 18:3(n-3), 18:1(n-9) and 22:5(n-6) appeared to be slowly metabolized by seminal monooxygenases, while 18:2(n-6) was converted to the w3 alcohol and to smaller amounts of the w2 alcohol (4:1). Together, the results indicate that the w3-hydroxylase and w3-epoxygenase enzyme(s) metabolize 20:4(n-6) and 20:5(n-3) almost exclusively to the w3(R) alcohol and the w3(R, S) epoxide, respectively, while longer and shorter fatty acids either are poor substrates or metabolized with a lesser degree of position specificity.     

Isolation of two novel E prostaglandins in human seminal fluid

cis-8,11,14,17-[1-14C]Eicosatetraenoic acid was incubated with microsomes of ram seminal vesicles and 1 mM glutathione for 3 min at 37 degrees C. The main metabolite was identified as 17,18-dehydroprostaglandin E1 by capillary column gas chromatography-mass spectrometry. Human seminal fluid was analyzed for the presence of 17,18-dehydroprostaglandin E1 and prostaglandin E3. Whereas prostaglandin E3 could be demonstrated by capillary gas chromatography-mass spectrometry, 17,18-dehydroprostaglandin E1 could not be found under these conditions. However, human seminal fluid contained two compounds with a similar polarity on reversed phase high performance liquid chromatography as 17,18-dehydroprostaglandin E1 and prostaglandin E3. The two compounds were identified as 18,19-dehydroprostaglandin E1 and 18,19-dehydroprostaglandin E2 by gas chromatography-mass spectrometry, by UV analysis after conversion to the corresponding prostaglandin B compounds, and by ozonolysis. The amount of each of the two prostaglandins in human seminal fluid seemed to be in the same order of magnitude as the amount of prostaglandin E3.     

On the metabolism of epoxyeicosatrienoic acids by ram seminal vesicles: isolation of 5(6)epoxy-prostaglandin F1 alpha

cis-5(6)Epoxy- and cis-14(15)epoxyeicosatrienoic acid are formed from arachidonic acid by monooxygenases. 5(6)Epoxyeicosatrienoic acid is metabolized by fatty acid cyclooxygenase of ram seminal vesicles and the major products were recently identified as 5(6)epoxy-PGE1 and two stereoisomers of 5-hydroxy-PGI1. The two isomers were likely formed from an unstable intermediate, 5(6)epoxy-PGF1 alpha. The isolation of 5(6)epoxy-PGF1 alpha is described here and 14(15)epoxyeicosatrienoic acid is shown to inhibit fatty acid cyclooxygenase of ram seminal vesicles, albeit less potently than eicosatetraynoic acid (IC50 0.18 and 0.05 mM, respectively).     

Identification and biological activity of a novel natural prostagladin, 5,6-dihydro-prostaglandin E3

A novel natural E-prostaglandin was detected by HPLC among the endogenous prostaglandins extracted from ram seminal vesicles. The corresponding precursor — all-cis-eicosa-8,11,14,17-tetraenoic acid was isolated from bovine liver lipids and the preparative biosynthesis with the microsomal fraction of ram seminal vesicles was performed. The isolated product was purified by HPLC and identified by GC-MS as 5,6-dihydro-PGE₃. The results of in vitro testss demonstrate that 5,6-dihydro-PGE₃ is 14 times less active uterine stimulant than PGE₁, at the same time retaining 75% of the anti-aggregatory potency of PGE₁. Thus, 5,6-dihydro-PGE₃ meets the requirements of a selective antithrombotic agent more than PGE₁.     

Identification of 6-ketoprostaglandin F1alpha formed from arachidonic acid in bovine seminal vesicles.

The prostaglandin synthesizing system in bovine seminal vesicles was characterized by a radiometric assay. Two main products were formed from [1-14C]-arachidonic acid, and their structures were confirmed by mass spectrometry. The less polar product was identical with prostaglandin E2 and the more polar one was identical with a new prostaglandin, i.e., 6-ketoprostaglandin F1alpha.     

Metabolism of 5,6-epoxyeicosatrienoic acid by the human platelet. Formation of novel thromboxane analogs.

Radiolabeled cis-(+-)-5,6-epoxyeicosatrienoic acid (5(6)-EpETrE) was incubated with a suspension of isolated human platelets in order to study its metabolic fate. The epoxide slowly disappeared from the suspension and was completely metabolized within 30 min. After extraction and analysis by reverse-phase high performance liquid chromatography, seven metabolites were found. Addition of either indomethacin (0.01 mM, cyclooxygenase inhibitor) or BW755C (0.1 mM, cyclooxygenase/lipoxygenase inhibitor) to the incubations blocked the formation of four and six metabolites, respectively, 1,2-Epoxy-3,3,3-trichloropropane (inhibitor of microsomal epoxide hydrolase) failed to inhibit the formation of 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETrE), a hydrolysis product of the precursor 5(6)-EpETrE. The metabolites were characterized by UV spectroscopy, negative ion chemical ionization liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry and, in one instance, coelution with synthetic standard. Three primary platelet metabolites were structurally determined to be 5,6-epoxy-12-hydroxyeicosatrienoic acid, 5,6-epoxy-12-hydroxyheptadecadienoic acid, and a unique bicyclic metabolite, 5-hydroxy-6,9-epoxy-thromboxane B1, which originated from intramolecular hydrolysis of 5,6-epoxythromboxane-B1. This thromboxane analog was partially separated into stereoisomers and coeluted with the racemic synthetic standard in gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Three other metabolites were characterized as 5,6,12-trihydroxyeicosatrienoic acid, 5,6,12-trihydroxyheptadecadienoic acid, and 5,6-dihydroxythromboxane-B1, and resulted from the hydrolysis of the corresponding epoxides rather than from the metabolism of 5,6-DiHETrE. The latter was not metabolized by platelet cyclooxygenase or lipoxygenase. The biosynthesis of two cyclooxygenase metabolites indicated the formation of unstable 5,6-epoxythromboxane-A1 as an intermediate precursor. Platelet aggregation was not induced by 5(6)-EpETrE, although responsiveness to arachidonic acid was reduced following preincubation with the epoxide. The platelet metabolites of 5(6)-EpETrE might be useful in assessing its in vivo production in humans.     

Oxygenation of 5,8,11-eicosatrienoic acid by prostaglandin H synthase-2 of ovine placental cotyledons: isolation of 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids

Prostaglandin H synthase-1 of ram vesicular glands metabolises 5,8,11-eicosatrienoic (Mead) acid to 13R-hydroxy-5,8,11-eicosatrienoic and to 11R-hydroxy-5,8,12-eicosatrienoic in a 5:1 ration. We wanted to determine the metabolism of this fatty acid by prostaglandin H synthase-2. Western blot showed that microsomes of sheep and rabbit placental cotyledons contained prostaglandin H synthase-2, while prostaglandin H synthase-1 could not be detected. Microsomes of sheep cotyledons metabolised [1-¹⁴C]5,8,11-eicosatrienoic acid to many polar metabolites and diclofenac (0.05 mM) inhibited the biosynthesis. The two major metabolites were identified as 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids. They were formed in a ratio of 3:2, which was not changed by aspirin (2 mM). 5,8,11-Eicosatrienoic acid is likely oxygenated by removal of the pro-S hydrogen at C-13 and insertion of molecular oxygen at either C-13 or C-11, which is followed by reduction of the peroxy derivatives to 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids, respectively. Prostaglandin H synthase-1 and -2 oxygenate 5,8,11-eicosatrienoic acid only slowly compared with arachidonic acid.     

Characterization of prostaglandin E2 20-hydroxylase of sheep vesicular glands

The microsomal fraction of homogenates of the sheep vesicular glands, supplemented with 1 mM NADPH, metabolized 0.2 mM prostaglandin E2 to 20-hydroxyprostaglandin E2 at a rate of 76 +/- 9 pmol/min per mg of protein (with a Km of about 0.1 mM and a Vmax of about 0.1 nmol/min per mg of protein). Prostaglandin E1 was metabolized at a rate of only 8.5% of that of prostaglandin E2. The metabolism of prostaglandin E2 was decreased by 66% using 1 mM NADH instead of NADPH. alpha-Naphthoflavone (50 microM) and carbon monoxide inhibited the 20-hydroxylase by more than 60%, while 1 mM beta-diethylaminoethyl-2,2-diphenyl-pentanoate and 1 mM metyrapone inhibited it by less than 50%. The enzyme catalyzed the incorporation of atmospheric oxygen into the substrate. The findings suggest that the 20-hydroxylase could be a cytochrome P-450. The 20-hydroxylase could not be detected in vesicular glands of five rams 3 weeks after castration. The function of the enzyme is presumably to create the high level of 20-hydroxyprostaglandin E compounds in ram semen.     

Biosynthesis and metabolism of prostaglandins in chick epiphyseal cartilage.

Microsomal fractions of cells isolated from chick epiphyseal cartilage catalyzed the synthesis of prostaglandins from radiolabeled delta8,11,14-eicosatrienoic and from arachidonic acids. In addition, the microsomal supernatants contained both 15-hydroxyprostaglandin dehydrogenase and prostaglandin 15-keto delta13,14-reductase activities. Two major classes of prostaglandins (E and F) were synthesized; however, a major product which chromatographically behaves as PGA was also isolated. Synthetase activities were analyzed for pH optima and response to known stimulators and inhibitors of prostaglandin systhesis. The different activators had varying stimulatory effects on prostaglandin synthesis; the anti-inflammatory drugs were all strongly inhibitory. Synthetase activity in the growth plate was highest in the zone of hypertrophy, declining substantially in the more heavily calcified regions. Degradative enzyme activities were highest in the zone of maturation and significantly lower in the adjacent hypertrophic zone. The net effect of these opposing activities would be to elevate prostaglandin levels at the zone of hypertrophy, a finding which suggests that prostaglandins may play a role in the modulation of epiphyseal cartilage metabolism.     

The metabolism of benz[a]anthracene and dibenz[a,h]anthracene and their 5,6-epoxy-5,6-dihydro derivatives by rat-liver homogenates

1. Benz[a]anthracene was hydroxylated by rat-liver homogenates on the 3,4-,5,6- or 8,9-bond to yield phenols and dihydrodihydroxy compounds. Metabolic action at the 7- and 12-positions was also detected. 5,6-Epoxy-5,6-dihydrobenzanthracene was converted into a phenol that is probably 5-hydroxybenzanthracene and 5,6-dihydro-5,6-dihydroxybenzanthracene. Both substrates yielded a product that is probably S-(5,6-dihydro-6-hydroxy-5-benzanthracenyl)glutathione. 2. Dibenz[a,h]anthracene was hydroxylated by rat-liver homogenates to yield products that are probably 3- and 4-hydroxydibenzanthracene, 1,2-dihydro-1,2-dihydroxydibenzanthracene, 3,4-dihydro-3,4-dihydroxydibenzanthracene and 5,6-dihydro-5,6-dihydroxydibenzanthracene. There was no evidence for metabolic action at the 7- and 14-positions. 5,6-Epoxy-5,6-dihydrodibenzanthracene was converted into a phenol that is probably 5-hydroxydibenzanthracene and 5,6-dihydro-5,6-dihydroxydibenzanthracene. Both substrates yielded a glutathione conjugate that is probably S-(5,6-dihydro-6-hydroxy-5-dibenzanthracenyl)glutathione. 3. The synthesis of 5,6-epoxy-5,6-dihydrodibenzanthracene is described and the reactions of this epoxide and 5,6-epoxy-5,6-dihydrobenzanthracene with water and thiols have been investigated. 4. The oxidation of dibenzanthracene in the ascorbic acid-Fe(2+) ion-oxygen model system is described.     

Enzymatic hydration of leukotriene A4. Purification and characterization of a novel epoxide hydrolase from human erythrocytes

Human erythrocytes contained a soluble cytosolic epoxide hydrolase for stereospecific enzymatic hydration of leukotriene A4 into leukotriene B4. The enzyme was purified 1100-fold, to apparent electrophoretic homogeneity, by conventional DEAE-Sephacel fractionation followed by high performance anion exchange and chromatofocusing procedures. Its characteristics include a molecular weight of 54,000 +/- 1,000, an isoelectric point 4.9 +/- 0.2, a Km apparent from 7 to 36 microM for enzymatic hydration of leukotriene A4, and a pH optimum ranging from 7 to 8. The enzyme was partially inactivated by its initial exposure to leukotriene A4. There was slow but detectable enzymatic hydration (pmol/min/mg) of certain arachidonic acid epoxides including (+/-)-14,15-oxido-5,8-11-eicosatrienoic acid and (+/-)-11,12-oxido-5,8,14-eicosatrienoic acid, but not others, including 5,6-oxido-8,11,14-eicosatrienoic acid. Human erythrocyte epoxide hydrolase did not hydrate either styrene oxide or trans-stilbene oxide. In terms of its physical properties and substrate preference for leukotriene A4, the erythrocyte enzyme differs from previously described versions of epoxide hydrolase. Human erythrocytes represent a novel source for an extrahepatic, cytosolic epoxide hydrolase with a potential physiological role.     

Biosynthesis and metabolism of prostaglandins in chick epiphyseal cartilage

Microsomal fractions of cells isolated from chick epiphyseal cartilage catalyzed the synthesis of prostaglandins from radiolabeled Δ^8,11,14-eicosatrienoic and from archidonic acids. In addition, the microsomal supernatants contained both 15-hydroxyprostaglandin dehydrogenase and prostaglandin 15-keto Δ^13,14-reductase activities. Two major classes of prostaglandins (E and F) were synthesized; however, a major product which chromatographically behaves as PGA was also isolated. Synthetase activities were analyzed for pH optima and response to known stimulators and inhibitors of prostaglandin synthesis. The different activators had varying stimulatory effects on prostaglandin synthesis; the anti-inflammatory drugs were all strongl inhibitory. Synthetase activity in the growth plate was highest in the zone of hypertrophy, declining substantially in the more heavily calcified regions. Degradative enzyme activities were highest in the zone of maturation and significantly lower in the adjacent hypertrophic zone. The net effect of these opposing activities would be to elevate prostaglandin levels at the zone of hypertrophy, a finding which suggests that prostaglandins may play a role in the modulation of epiphyseal cartillage metabolism.     

The formation of styrene glutathione adducts catalyzed by prostaglandin H synthase. A possible new mechanism for the formation of glutathione conjugates

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.     

Prostaglandin endoperoxides 21. Covalent binding of levuglandin E2 with proteins

Levuglandin E2 (LGE2), a gamma-ketoaldehyde produced by rearrangement of the prostaglandin endoperoxide PGH2 under the aqueous conditions of its biosynthesis, binds covalently with ram seminal vesicle microsomes. Totally synthetic 5,6-ditritio-LGE2 was prepared and used to determine that rapid covalent binding of LGE2 (initially 800 microM) occurs with 6.4 microM bovine serum albumin (greater than 10 equiv within 1 min) which approaches saturation (approximately 16 equiv) after 40 min at 37 degrees C.     

Spectral intermediates of prostaglandin hydroperoxidase

Microsomes from ram seminal vesicles or purified prostaglandin H synthase supplemented with either arachidonic acid or prostaglandin G2 formed an unstable spectral intermediate with maxima at 430 nm, 525 nm and 555 nm and minima at 410 nm, 490 nm and 630 nm. At -15 degrees C the band at 430 nm disappeared within 4 min whereas the trough at 410 nm increased three fold. At higher temperatures (10-37 degrees C) spectral complex formation and decay were observed in less than 1 s. An apparent KS-value of about 3 microM was determined for the titration of purified prostaglandin synthase with prostaglandin G2 at -20 degrees C. Substrates for cooxidation reactions of prostaglandin synthase such as phenol, hydroquinone and reduced glutathione as well as the peroxidase inhibitors cyanide and azide inhibited the prostaglandin G2-induced spectral complex formation. The oxene donor iodosobenzene and hydrogen peroxide formed a spectral intermediate analogous to the complex observed with prostaglandin G2 or arachidonic acid in ram seminal vesicle microsomes as well as with the purified prostaglandin synthase. These results are interpreted as the formation of a ferryl-oxo complex (FeO)3+ of the heme of prostaglandin synthase with prostaglandin G2 analogous to the formation of compound I of horseradish peroxidase.