Prostaglandin D2 formation and characterization of its synthetases in various tissues of adult rats

When the amounts of primary prostaglandins formed from endogenous arachidonic acid were determined in homogenates of various tissues of adult rats, prostaglandin D2 was the major prostaglandin found in most tissues. It was formed actively in the spleen (3100 ng/g tissue/5 min at 25 degrees C), intestine (2600), bone marrow (2400), lung (1100), and stomach (630); moderately in the epididymis, skin, thymus, and brain (140-340); and weakly in other tissues (less than 100). Addition of exogenous arachidonic acid (1 mM) accelerated the formation of prostaglandin D2 in all tissues as follows: spleen (15,000); bone marrow, intestine, thymus, liver, and lung (1600-5200); stomach, adrenal gland, epididymis, brain, salivary gland, skin, spinal cord, and seminal vesicle (380-1000); and other tissues (80-310). The activity of prostaglandin D synthetase (prostaglandin-H2 D-isomerase) was detected in 100,000g supernatants of almost all tissues. As judged by glutathione requirement for the reaction, inhibition of the activity by 1-chloro-2,4-dinitrobenzene, and immunotitration or immunoabsorption analyses with specific antibodies, the enzyme in the epididymis, brain, and spinal cord (1.8-9.2 nmol/min/mg protein) was glutathione-independent prostaglandin D synthetase (Y. Urade, N. Fujimoto, and O. Hayaishi (1985) J. Biol. Chem. 260, 12410-12415). The enzyme in the spleen, thymus, bone marrow, intestine, skin, and stomach (2.0-57.1) was glutathione-requiring prostaglandin D synthetase (Y. Urade, N. Fujimoto, M. Ujihara, and O. Hayaishi (1987) J. Biol. Chem. 262, 3820-3825). The activity in the kidney and testis (3.7-4.5) was catalyzed by glutathione S-transferase. The activity in the liver, lung, adrenal gland, salivary gland, heart, pancreas, and muscle (0.6-5.1) was due to both the glutathione-requiring synthetase and the transferase.     

The subunit structure of a major glutathione S-transferase form, MT, in rat testis. Evidence for a heterodimer consisting of subunits with different isoelectric points

A major glutathione S-transferase form (pI 5.7) in rat testis (MT) purified by S-hexyl-glutathione affinity chromatography, followed by chromatofocusing, showed two polypeptide of pI 6.7 (Yn1) and 6.0 (Yn2), having apparently the same molecular mass of 26 kDa on two-dimensional gel electrophoresis. Rechromatofocusing of the MT preparation after 4 M guanidine hydrochloride treatment revealed two additional protein peaks (pI 6.2 and 5.4). These were identified as the two homodimers consisting of the subunits of MT, Yn1Yn1 and Yn2Yn2, respectively. Furthermore, MT could be reconstituted from Yn1Yn1 and Yn2Yn2. These results indicate that MT is a heterodimer, Yn1Yn2, consisting of subunits with very similar molecular masses but different isoelectric points. The Yn1Yn1 form had glutathione S-transferase activities towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene. However, the Yn2Yn2 form had no activity towards any of the substrates examined. N-terminal amino acid sequences of subunits Yn1 and Yn2 revealed differences at two positions in the first 20 residues; the amino acid compositions of these subunits were also similar but not identical, indicating that these two subunits are different in the primary structure. Subunits Yn1 and Yn2 are immunologically related to each other and also to subunits 3 (Yb1) and 4 (Yb2) but they are not identical. These four subunits also showed a high degree of similarity in N-terminal amino acid sequences. Subunits Yn1 and Yn2 seem to belong to the rat GST 3-4 family or class mu. Subunits Yn1 and 4 can make a heterodimer, which is detectable not only in rat testis, but also in the heart, kidney and lung. The Yn1Yn1 form was not detected in the testis, but is present in rat brain [Tsuchida et al. (1987) Eur. J. Biochem. 170, 159-164]. The Yn2Yn2 form seemed to differ from GST 5-5 and may be a new form of rat glutathione S-transferase.     

Purification of a new acidic glutathione S-transferase, GST-Yn1Yn1, with a high leukotriene-C4 synthase activity from rat brain

A new acidic form of glutathione S-transferase (GST, pI 6.2) was purified from rat brain by S-hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20-25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (MT or 6-6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the MT in isoelectric point, activity towards 1,2-dichloro-4-nitrobenzene and immunological properties. On two-dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn1) of two spots (Yn1 and Yn2) of the testis GST-MT. Therefore, the brain acidic form is a homodimer, and named GST-Yn1Yn1. The activity was inhibited by sulfasalazine, an inhibitor of leukotriene-C4 synthase. This form (GST-Yn1Yn1) showed the highest leukotriene-C4 synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The Km values for leukotriene A4 and glutathione were 26 microM and 3.5 mM respectively. A major GST form of rat brain, occupying about 40% of the total activity, was identical with GST-P (7-7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST-P also showed the significant leukotriene-C4 synthase activity, 67.2 nmol/mg protein in 5 min, but the Km for leukotriene A4 was 100 microM, fourfold higher than that of GST-Yn1 Yn1. These results suggest that mainly GST-Yn1 Yn1 may be involved in leukotriene-C4 synthesis in rat brain.     

The 1- and 2-electron oxidation of acetaminophen catalyzed by prostaglandin H synthase

Purified and microsomal preparations of prostaglandin H synthase catalyzed the arachidonic acid-dependent polymerization of acetaminophen and, in the presence of GSH, catalyzed the formation of 3-(glutathion-S-yl)acetaminophen. The formation of these products was inhibited by indomethacin and by purging reaction mixtures with argon. When H2O2 replaced arachidonic acid, neither indomethacin nor argon purging inhibited product formation. These results suggest that the peroxidase activity of prostaglandin H synthase catalyzed the oxidation of acetaminophen. Addition of GSH to reaction mixtures decreased acetaminophen polymerization; however, 3-(glutathion-S-yl)acetaminophen formation was maximal with 40 microM GSH, and higher concentrations of GSH did not substantially alter its formation. In the presence of GSH, either ascorbic acid or NADPH decreased polymerization by greater than 97% while 3-(glutathion-S-yl)acetaminophen formation was still observed. These data suggest that polymers and conjugates were formed by two different pathways. Since polymerization of acetaminophen involves radical termination of N-acetyl-p-benzosemiquinone imine whereas 3-(glutathion-S-yl)acetaminophen is formed by conjugation of N-acetyl-p-benzoquinone imine with GSH, the data suggest that prostaglandin H synthase catalyzed both the overall 1- and 2-electron oxidation of acetaminophen.     

Enzymatic formation of prostaglandin F2 alpha from prostaglandin H2 and D2. Purification and properties of prostaglandin F synthetase from bovine lung

Prostaglandin F synthetase from bovine lung was purified 540-fold to apparent homogeneity, as assessed by polyacrylamide gel electrophoreses and ultracentrifugation. The purified enzyme proved to be a monomeric protein with a molecular weight of about 30,500. The enzyme catalyzed not only the reduction of the 11-keto group of prostaglandin D2 but also the reduction of 9,11-endoperoxide of prostaglandin H2 and various carbonyl compounds (e.g. phenanthrenequinone). Experiments using column chromatography, polyacrylamide gel electrophoreses, immunotitration using antibody against the purified enzyme, and heat treatment indicated that three enzyme activities resided in a single protein. Although phenanthrenequinone and prostaglandin D2 competitively inhibited the prostaglandin D2 and phenanthrenequinone reductase activities, respectively, these two substrates were all but ineffective on the prostaglandin H2 (at the Km value) reductase activity up to 14-fold of those Km values. These results suggest that a single enzyme protein purified from the bovine lung catalyzes the reduction of prostaglandin D2, prostaglandin H2, and various carbonyl compounds and that prostaglandin D2 and prostaglandin H2 are metabolized at two different active sites, yielding prostaglandin F2 alpha as the reaction product.     

Purification and characterization of glutathione S-transferases P, S and N. Isolation from rat liver of Yb1 Yn protein, the existence of which was predicted by subunit hybridization in vitro.

The glutathione S-transferases are dimeric proteins and comprise subunits of Mr 25 500 (Ya), 26 500 (Yn), 27 000 (Yb1 and Yb2) and 28 500 (Yc). Enzymes containing Ya and/or Yc subunits have been isolated as have forms containing binary combinations of Yn, Yb1 and Yb2 subunits. To date only one enzyme, transferase S, has been described that is a YbYn heterodimer [Hayes & Chalmers (1983) Biochem. J. 215, 581-588]; the identity of the Yb monomer found in transferase S has not been reported previously. The identification and isolation of a YnYn dimer (transferase N) from rat testis is now described. This has enabled structural and functional comparisons to be made between Yb1, Yb2 and Yn monomers. Reversible dissociation experiments between the YnYn and Yb1Yb1 homodimers and between the YnYn and Yb2Yb2 homodimers demonstrated that Yn monomers can hybridize with both Yb1 and Yb2 monomers. Reversible dissociation of transferases N and C (Yb1Yb2) showed that both Yb1 and Yb2 monomers can hybridize with Yn monomers under competitive conditions. The hydridization data suggest that transferase S represents the Yb2Yn subunit combination. A knowledge of the elution position from chromatofocusing columns of the Yb1Yn hybrid that was formed in vitro enabled a purification scheme to be devised for an enzyme from rat liver (transferase P) believed to consist of Yb1Yn subunits. A comparison of the chromatographic behaviour of the YnYn, Yb1Yb1 and Yb2Yb2 dimers on chromatofocusing and hydroxyapatite columns with the behaviour of transferases P and S on the same matrices suggests these two enzymes may be identified as the Yb1Yn and Yb2Yn dimers respectively. The catalytic activities and the inhibitory effects of non-substrate ligands on transferases P and S are significantly different and again suggest they comprise Yb1 and Yn subunits and Yb2 and Yn subunits respectively; transferase P exhibits a 6-fold higher specific activity for 1,2-dichloro-4-nitrobenzene than does transferase S, whereas, conversely, transferase S possesses a 9-fold higher specific activity for trans-4-phenylbut-3-en-2-one than does transferase P. The quaternary structure of transferases P and S was verified by using peptide mapping and 'Western blotting' techniques.     

Biochemical and immunological characterization of rat spleen prostaglandin D synthetase

Rat spleen prostaglandin D synthetase (Christ-Hazelhof, E., and Nugteren, D. H. (1979) Biochim. Biophys. Acta 572, 43-51) is very similar to rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi O. (1985) J. Biol. Chem. 260, 12410-12415) as judged by their pI (4.7-5.2), Mr (26,000-27,000), and self-inactivation during the isomerase reaction from prostaglandin H2 to prostaglandin D2. However, the amino acid compositions of these two enzymes were quite different. Furthermore, the spleen enzyme was associated with the glutathione S-transferase activity, differing from the brain enzyme. The synthetase and transferase activities of the spleen enzyme showed almost identical pH and glutathione dependencies, the optimum pH = 8.0 and Km for glutathione = 300 microM. The Km values for prostaglandin H2 and 1-chloro-2,4-dinitrobenzene (a substrate for the transferase) were about 200 microM and 5 mM, respectively. The synthetase activity was dose-dependently inhibited by 1-chloro-2,4-dinitrobenzene (IC50: approximately 5 mM) and more strongly by nonsubstrate ligands, such as bilirubin and indocyanine green (IC50: 150 and 2 microM, respectively). Both the synthetase and transferase activities of the purified enzyme dose-dependently decreased and showed identical immunotitration curves by incubation with antibody against this enzyme, but remained unchanged when treated with antibody against the brain enzyme. The antibody specific for the spleen enzyme absorbed almost all of the synthetase activity and about 10% of the transferase activity in the spleen, but not the transferase activity in the liver, heart, and testis. These results show that the two types of prostaglandin D synthetase are similar but different enzymes and that the spleen enzyme is a unique glutathione S-transferase differing from other isozymes and their subunits reported previously.     

Enzymatic conversion of prostaglandin H2 to prostaglandin F2 alpha by aldehyde reductase from human liver: comparison to the prostaglandin F synthetase from bovine lung

The primary structure of prostaglandin (PG) F synthetase from bovine lung shows 62% similarity with that of human liver aldehyde reductase (EC 1.1.1.2) (Watanabe, K., Fujii, Y., Nakayama, K., Ohkubo, H., Kuramitsu, S., Kagamiyama, H., Nakanishi, S., and Hayaishi, O. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 11-15). We therefore purified human liver aldehyde reductase to homogeneity and compared the immunological and catalytic properties of aldehyde reductase and PGF synthetase. Although both enzymes belong to a group of aldoketoreductases and their molecular weights are essentially identical, aldehyde reductase had no cross-reactivity to anti-PGF synthetase antiserum. Furthermore, there was a difference in the substrate specificity for reduction of PGs between the two enzymes. Aldehyde reductase catalyzed the reduction of PGJ2, delta 12-PGJ2, PGH2, or PGA2, but not that of PGB2, PGD2, or PGE2, whereas PGF synthetase reduced PGD2. The optimum pH, Km value for PGH2, and the turnover number were 6.5, 100 microM, and 3.1 min-1, respectively. The PGH2 9,11-endoperoxide reductase activity of aldehyde reductase was not affected in the presence of a substrate such as p-nitrobenzaldehyde, DL-glyceraldehyde, or 9,10-phenanthrenequinone, suggesting that PGH2 9,11-endoperoxide and other substrates are reduced at different active site(s). The reaction product formed from PGH2 by this enzyme was identified as PGF2 alpha by gas chromatography/mass spectrometry. These results suggest that aldehyde reductase is not exactly identical to PGF synthetase in terms of its immunological property and substrate specificity for PGs, but that this enzyme is also involved in the direct conversion of PGH2 to PGF2 alpha similar to PGF synthetase.     

Purification of the thromboxane A2/prostaglandin H2 receptor from human blood platelets

S-145 (5Z-7-(3-endo-phenylsulfonylamino-(2.2.1.)-bicyclohept -2-exo-yl) heptenoic acid) is a potent and selective antagonist for thromboxane A2/prostaglandin H2 receptor. Using this compound as an immobilized ligand for affinity chromatography and [3H]S-145 as a radioligand, we have purified the thromboxane A2/prostaglandin H2 receptor from the membranes of human blood platelets. The purification procedures consisted of solubilization of the receptor with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), affinity chromatographies on columns of S-145 affinity gel, wheat germ agglutinin agarose and red agarose, and repeated gel filtration high performance liquid chromatography on a TSK gel G-3000SW column. On the second gel filtration high performance liquid chromatography, the [3H]S-145 binding activity was eluted as a symmetrical peak which overlapped exactly with a peak of ultraviolet absorption at 280 nm. By these procedures, the receptor was purified about 8700-fold from the solubilized extract with a recovery of 6%. The final preparation showed a broad protein band at Mr 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maximally bound 19.2 nmol of [3H]S-145/mg protein with a Kd of 29.8 nM. The [3H]S-145 binding to the purified receptor was specifically displaced by several thromboxane A2/prostaglandin H2 analogues.     

Synthesis and inhibitory activity against COX-2 catalyzed prostaglandin production of chrysin derivatives

A series of chrysin derivatives were prepared and evaluated for their inhibitory activities of cyclooxygenase-2 catalyzed prostaglandin production. Chrysin derivatives were prepared from 2-hydroxyacetophenone, 2,4-dihydroxyacetophenone and 2,6-dihydroxyacetophenone in 2 to 4 steps, respectively. Methxoylated chrysin derivatives were converted to the corresponding hydroxylated chrysin derivatives by the reaction with BBr(3) in good yields. The inhibitory activity of the chrysin derivatives against prostaglandin production from lipopolysaccharide-treated RAW 264.7 cells was measured. We found that chrysin derivatives with 3',4'-dichloro substituents (5e, 6e and 7e) exhibited good inhibitory activity of prostaglandin production.     

Solubilization and partial purification of prostaglandin endoperoxide synthetase of rabbit kidney medulla.

The microsomes of rabbit kidney medulla converted arachidonic acid into prostaglandin E2 in the presence of hemoglobin, tryptophan and glutathione as activators. When themicrosomal suspension was treated with 1% Tween 20, a solubilized enzyme was obtained which catalyzed the conversion of arachidonic acid to prostaglandins G2 and H2. The solubilized enzyme was adsorbed to and then eluted from an omega-aminooctyl Sepharose 4B column, resulting in about 10-fold purification over the microsomes. The partially purified enzyme produced predominantly prostaglandin G2 in the presence of hemoglobin, while prostaglandin H2 was produced in the presence of both hemoglobin and tryptophan. The stimulation of prostaglandin endoperoxide formation was also observed with other heme and aromatic compounds. Prostaglandin H2 synthesis was inhibited by a variety of compounds including non-steroidal anti-inflammatory drugs, thiol compounds and prostaglandin analogues with a thiol group(s).     

9 alpha,11 beta-prostaglandin F2 formation in various bovine tissues. Different isozymes of prostaglandin D2 11-ketoreductase, contribution of prostaglandin F synthetase and its cellular localization

9 alpha,11 beta-prostaglandin F2 was formed from prostaglandin D2 by its 11-ketoreductases in 100,000 x g supernatants of various bovine tissues in the presence of an NADPH-generating system. The reductase activities were high in liver (51.09 nmol/h/mg of protein), lung (24.99), and spleen (14.20); moderate in heart and pancreas (3.09-3.61); weak in stomach, intestine, colon, kidney, uterus, adrenal gland, and thymus (0.11-2.63); and undetectable in brain, retina, carotid artery, and blood (less than 0.10). No formation of prostaglandin F2 alpha from prostaglandin D2 was detected in all tissues. In immunotitration analyses with a polyclonal antibody specific for prostaglandin F synthetase, the reductase activities in lung and spleen showed identical titration curves to that of the purified synthetase and decreased to less than 15% of the initial activity under the condition of antibody excess. Prostaglandin F synthetase-immunoreactive protein in these two tissues showed peptide fingerprints identical to that of the purified enzyme after partial digestion with Staphylococcus aureus V8 protease. The antibody was partially cross-reactive to the reductase in liver (about 20% of that to the synthetase) but not to the reductase(s) in other tissues. The Km value for prostaglandin D2 of the reductase activity was the same in lung and spleen as that of the purified prostaglandin F synthetase (120 microM) but differed in liver (6 microM), heart, and pancreas (15 microM). The predominant distribution of prostaglandin F synthetase in lung and spleen was confirmed by radioimmunoassay (2.8 and 1.0 micrograms/mg protein, respectively) and Northern blot analyses. In immunoperoxidase staining, this enzyme was localized in alveolar interstitial cells and nonciliated epithelial cells in lung, histiocytes and/or dendritic cells in spleen, and a few interstitial cells in kidney and adrenal cortex.     

Inhibition of prostaglandin biosynthesis by c-5,c-8,c-11-eicosatrienoic acid.

The enzyme prostaglandin H leads to E-isomerase (EC 5.3.99.3), which is present in sheep vesicular gland and needs glutathione as cofactor, is inhibited by c-5,c-8,c-11-eicosatrienoic acid, the fatty acid accumulating during essential fatty acid deficiency. The EFA-deficiency syndrome can partly be explained from a prostaglandin deficiency caused by lack of precursors. The present finding indicates that 5,8,11-eicosatrienoic acid could well be an additional factor in modifying the symptoms of EFA-deficiency.     

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.     

Thiopental and ether anaesthesia decrease the prostaglandin receptor density of the rat liver.

Male Sprague-Dawley rats were killed either by decapitation or during anaesthesia with thiopental or diethylether by aortectomy. Livers were removed and liver plasma membranes were prepared using standard techniques. Direct binding experiments with 3H-PGE1 and 3H-iloprost revealed heterogeneity of the binding sites (high and low affinity binding sites), whereas 3H-PGE2 demonstrated only high affinity binding to the liver. The highest binding capacity for all radioligands was found for livers after decapitation. Livers obtained during anaesthesia showed a significantly (p less than 0.05 to p less than 0.001) lower binding capacity and binding affinity for 3H-PGE1, 3H-PGE2 and 3H-iloprost. The reduction in binding activity was more pronounced in livers obtained during inhalation than thiopental anaesthesia. Specific binding amounted to 82.1 +/- 7% for 3H-PGE2, 75.3 +/- 9% for 3H-PGE1 and 78.9 +/- 8% for iloprost in livers obtained after decapitation. In livers obtained during anaesthesia specific prostaglandin binding was significantly (p less than 0.01) decreased, again being more pronounced during inhalation than thiopental anaesthesia. These results suggest that some anaesthetics interfere with prostaglandin receptors of the liver.     

Stimulation of prostaglandin E2 production by phorbol esters and epidermal growth factor in porcine thyroid cells

Effects of phorbol esters and epidermal growth factor (EGF) on prostaglandin E2 production by cultured porcine thyroid cells were examined. Both phorbol 12-myristate 13-acetate (PMA) and EGF stimulated prostaglandin E2 production by the cells in dose related fashion. PMA stimulated prostaglandin E2 production over fifty-fold with the dose of 10(-7) M compared with control. EGF (10(-7) M) also stimulated it about ten-fold. The ED50 values of PMA and EGF were respectively around 1 X 10(-9) M and 5 X 10(-10) M. Thyroid stimulating hormone (TSH), however, did not stimulate prostaglandin E2 production from 1 to 24-h incubation. The release of radioactivity from [3H]-arachidonic acid prelabeled cells was also stimulated by PMA and EGF, but not by TSH. These results indicate that both PMA and EGF are potent stimulators of prostaglandin E2 production, associated with the activity to stimulate arachidonic acid release in porcine thyroid cells.     

Xenobiotic-metabolizing cytochromes P450 convert prostaglandin endoperoxide to hydroxyheptadecatrienoic acid and the mutagen, malondialdehyde

Cyclooxygenases catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides. Cyclooxygenase-2- and the xenobiotic-metabolizing cytochrome P450s 1A and 3A are all aberrantly expressed during colorectal carcinogenesis. To probe for a role of P450s in prostaglandin endoperoxide metabolism, we studied the 12-hydroxyheptadecatrienoate (HHT)/malondialdehyde (MDA) synthase activity of human liver microsomes and purified P450s. We found that human liver microsomes have HHT/MDA synthase activity that is concentration-dependent and inhibited by the P450 inhibitors, ketoconazole and clotrimazole with IC(50) values of 1 and 0.4 microM, respectively. This activity does not require P450 reductase. HHT/MDA synthase activity was present in purified P450s but not in heme alone or other heme proteins. The catalytic activities of various purified P450s were determined by measuring rates of MDA production from prostaglandin endoperoxide. At 50 microM substrate, the catalytic activities of purified human P450s varied from 10 +/- 1 to 0.62 +/- 0.02 min(-1), 3A4 > 2E1 > 1A2. Oxabicycloheptane analogs of prostaglandin endoperoxide, U-44069 and U-46619, induced spectral changes in human P450 3A4 with K(s) values of 240 +/- 20 and 130 +/- 10 microM, respectively. These results suggest that co-expression of cyclooxygenase-2 and P450s in developing cancers may contribute to genomic instability due to production of the endogenous mutagen, MDA.     

Effect of prostaglandin F2alpha on gonadal carbohydrate metabolism of silkworm,Bombyx mori L

Effect of prostaglandin F2alpha (PGF2alpha) on carbohydrate accumulation in gonads of the multivoltine silkworm (hybrid: Xinhang x Keming) has been studied by means of topical application to larvae of the silkworm. Increased weights of larvae and reproduction organs, as well as carbohydrate metabolism in gonads of the silkworm, Bombyx mori L. was found after treatment with prostaglandin F2alpha. The increase in weight (larvae 21.9%, testis 28.9%, and ovary 33.3%) was associated with increases in the biomolecules (20-30%) and LDH and aldolase activity (18-25%). The results suggest that the accumulation of carbohydrates denotes a higher extent of utility of the energy sources in function of the testes and ovaries, and the routine application of prostaglandin F2alpha would be helpful in improving the reproductivity and egg quality of the silkworm.     

Mutation of a critical arginine in microsomal prostaglandin E synthase-1 shifts the isomerase activity to a reductase activity that converts prostaglandin H2 into prostaglandin F2alpha

Microsomal prostaglandin E synthase type 1 (mPGES-1) converts prostaglandin endoperoxides, generated from arachidonic acid by cyclooxygenases, into prostaglandin E2. This enzyme belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral membrane proteins, and because of its link to inflammatory conditions and preferential coupling to cyclooxygenase 2, it has received considerable attention as a drug target. Based on the high resolution crystal structure of human leukotriene C4 synthase, a model of mPGES-1 has been constructed in which the tripeptide co-substrate glutathione is bound in a horseshoe-shaped conformation with its thiol group positioned in close proximity to Arg-126. Mutation of Arg-126 into an Ala or Gln strongly reduces the enzyme's prostaglandin E synthase activity (85-95%), whereas mutation of a neighboring Arg-122 does not have any significant effect. Interestingly, R126A and R126Q mPGES-1 exhibit a novel, glutathione-dependent, reductase activity, which allows conversion of prostaglandin H2 into prostaglandin F2alpha. Our data show that Arg-126 is a catalytic residue in mPGES-1 and suggest that MAPEG enzymes share significant structural components of their active sites.     

Prostaglandin receptor-adenylate cyclase system in plasma membranes of rat liver and ascites hepatomas, and the effect of GTP upon it.

1. Adenylate cyclase in plasma membranes from rat liver was stimulated by prostaglandin E1, and to a lesser extent by prostaglandin E2. Prostaglandin F1alpha and A1 did not stimulate the cyclase. The prostaglandin E1-mediated activation was found to require GTP when the substrate ATP concentration was reduced from 3 mM to 0.3 mM in the reaction mixture. Adenylate cyclase of the plasma membranes from rat ascites hepatomas AH-130 and AH-7974 was not stimulated by prostaglandin E1 in the presence or the absence of GTP, although the basal activity of adenylate cyclase as well as its stimulation by GTP alone were similar to normal liver plasma membranes. 2. Liver plasma membranes were found to have two specific binders for [3H] prostaglandin E1 with dissociation constants of 17.6-10(-9) M and 13.6-10(8) M (37 degrees C) and one specific binder for [3H]prostaglandin F2alpha with a dissociation constant of 2.31-10(8) M (37 degrees C). The specific binders for prostaglandin E1 could not be detected in the hepatoma plasma membranes. 3. Binding of [3H] prostaglandin E1 to the liver plasma membranes was exchange by, GTP dGPT, GDP, ATP and GMP-P(N)P, but not by GMP, CGMP, DTTP, UTP or CTP. The increase in the binding of [3H] prostaglandin E1 was found to be due to the increased affinity of the specific binders to prostaglandin F2alpha was not affected by GTP. 4. GTP alone was found to increase V of adenylate cyclase of liver plasma membranes, while GTP plus prostaglandin E1 was found to decrease Km of adenylate cyclase in addition to the increase of V to a further extent.