Multiple molecular forms of prostaglandin 15-hydroxydehydrogenase and 9-ketoreductase in chicken kidney

Prostaglandin-15-hydroxydehydrogenase and prostaglandin-9-ketoreductase were purified from chicken kidney. Both enzymes exist in multiple forms as determined by isoelectric focusing. The dehydrogenases catalyze the transformation of the functional group at C-15 but not the functional group at C-9. The preferred cofactors in these reactions are NAD⁺ or NADH. The 9-ketoreductases catalyze the reversible transformation of the functional group at C-9 and also the oxidation or reduction of the C-15 functional group. The preferred cofactors are NADP⁺ or NADPH. Bradykinin does not affect the activities of any of the three prostaglandin 9-ketoreductases. Flavin mononucleotide and the flavonoid, quercetin, as well as indomethacin, ethacrynic acid, and furosemide, inhibit all three 9-ketoreductases. An inhibitor of 9-ketoreductase isolated from chicken breast muscle also inhibits the three separable reductases, but the pattern of inhibition of the reductase that focuses at pH 5.7 differs from that of the reductases focusing at pH 7.8 and 8.2.     

Multiple molecular forms of prostaglandin 15-hydroxydehydrogenase and 9-ketoreductase in chicken kidney.

Prostaglandin-15-hydroxydehydrogenase and prostaglandin-9-keto-reductase were purified from chicken kidney. Both enzymes exist in multiple forms as determined by isoelectric focusing. The dehydrogenases catalyze the transformation of the functional group at C-15 but not the functional group at C-9. The preferred cofactors in these reactions are NAD+ or NADH. The 9-ketoreductases catalyze the reversible transformation of the functional group at C-9 and also the oxidation or reduction of the C-15 functional group. The preferred cofactors are NADP+ or NADPH. Bradykinin does not affect the activities of any of the three prostaglandin 9-ketoreductases. Flavin mononucleotide and the flavonoid, quercetin, as well as indomethacin, ethacrynic acid, and furosemide, inhibit all three 9-ketoreductases. An inhibitor of 9-ketoreductase isolated from chicken breast muscle also inhibits the three separable reductases, but the pattern of inhibition of the reductase that focuses at pH 5.7 differs from that of the reductases focusing at pH 7.8 and 8.2.     

Partial purification and some properties of human erythrocyte prostaglandin 9-ketoreductase and 15-hydroxyprostaglandin dehydrogenase.

Human erythrocytes were found to contain two prostaglandin metabolizing enzymes: a prostaglandin E 9-ketoreductase catalyzing the reduction of prostaglandin E₂ to form prostaglandin F_2α and a 15-hydroxyprostaglandin dehydrogenase that catalyzes the oxidation of prostaglandin F_2α to form 15-ketoprostaglandin F_2α. Both enzymes are found in the cytoplasmic fraction of erythrocytes and both enzymes use the triphosphopyridine nucleotides as cofactors more effectively than the diphosphopyridine nucleotides. These two enzymes were partially purified from erythrocyte homogenates and some of their properties were studied.     

Prostaglandin E-2-9-ketoreductase in ovarian tissues.

The existence of the enzyme prostaglandin E-2-9-ketoreductase which can convert prostaglandin E-2 to prostaglandin F-2 alpha was indicated in experiments with pig and human ovarian tissues in vitro, using radioimmunoassay methods and a superfusion technique. Further studies involving radiotracer techniques demonstrated that the enzyme was localized in the high-speed (105 000 g) supernatant fraction of human, pig and rat luteal tissue and human stromal tissue. The enzyme was shown to be NADPH-dependent and its activity in luteal tissue increased in the order : pig less than human less than rat.     

Alterations of prostaglandin E2-9-ketoreductase activity in proliferating skin.

The activity of an NADPH-dependent PGE2-9-ketoreductase has been demonstrated in rat and human skin. This activity is localized in the high speed supernatant fraction, indicating the presence of an active PGE2-9-ketoreductase associated with the cytoplasmic fraction of the skin. Transformation of PGE2 into PGF2alpha is enhanced by skin specimens from psoriatic plaques and EFA-deficient rats, both characterized by excessive cellular proliferation and increased NADPH production. Incubations of the 105,000 g supernatant fractions from normal and EFA-deficient rats demonstrated that the activity of the PGE2-9-ketoreductase was elevated in high speed preparations from EFA-deficient rats. Results from these studies suggest that the increased activity of PGE2-9-ketoreductase observed in skin from human psoriatic plaques and EFA-deficient rats may be due in part to the increased generation of NADPH by these tissues and in part to alteration of the PGE2-9-ketoreductase by the excessive proliferation of the tissues.     

Demonstration of a thyrotropin-responsive prostaglandin E2-9-ketoreductase in bovine thyroid.

1. Prostaglandin E2-9-ketoreductase activity was demonstrated in bovine thyroid homogenates. 2. The enzyme requires reduced pyridine nucleotide and dialysis prior to assay for optimal activity. 3. The products of the reaction, NADP and prostaglandin F2alpha, inhibit enzyme activity. 4. Sigmoidal kinetics are observed when substrate concentration is plotted against enzyme velocity, indicative of an allosteric enzyme. 5. Thyrotropin increases enzyme activity in bovine thyroid slices. This increase is both hormone- and tissue-specific.     

Ovarian and uterine prostaglandin E2-9-ketoreductase activity in cyclic and pregnant ewes.

The regulation of luteal function in sheep appears to be dependent in part upon relative utero-ovarian concentrations of PGE2 and PGF2 alpha. Prostaglandin E2-9-ketoreductase converts PGE2 (a putative antiluteolysin) to PGF2 alpha. Enzymatic activity was measured in a cytosolic subcellular fraction of luteal and endometrial tissues collected on days 10, 13 and 16 of the estrous cycle or pregnancy. Respective days represented times before, during, and after the critical period for maternal recognition of pregnancy. Preparations of enzyme were incubated in the presence of tritiated PGE2. Radiolabeled PGF2 alpha (ie., product) was separated from PGE2 by gel filtration chromatography and quantified by liquid scintillation spectrometry. There were no significant differences due to time of tissue collection or pregnancy status in enzymatic activity of luteal tissues. Prostaglandin E2-9-ketoreductase activity isolated from endometria of open ewes was greater than their pregnant counterparts on days 13 and 16. Thus, the potential capacity of the ovine uterus to generate luteolytic PGF2 alpha from PGE2 substrate is elevated during an infertile estrous cycle.     

Prostaglandin E2-9-ketoreductase of rat testis.

A divalent metal dependent gluconolactonase has been isolated from porcine liver and purified to apparent homogeneity. Its molecular weight is estimated at 223,000 and that of the subunits is 37,200 as determined by gel electrophoresis. A K_m value of 6.2 mM was obtained at 27° in 50 mM tris HCl buffer. Gluconolactonase is specific for gluconolactone, and manganese is preferred over magnesium for maximum activity. The hepatic concentration of gluconolactonase is estimated to be 7.2 μmol of enzyme per kg of porcine liver, and a subcellular fractionation study indicates that this enzyme is located primarily within the cytosol.     

Selective stimulation of venous prostaglandin E 9-ketoreductase by bradykinin

The effects of bradykin on prostaglandin metabolism in canine mesenteric vessels were examined. Bradykinin stimulated microsomal prostaglandin synthesis in both artery and vein; this stimulation was more pronounced when [¹⁴C]phosphatidylcholine rather than [¹⁴C]arachidonate was used as the substrate for prostaglandin synthetase. This suggested that bradykinin enhanced a membrane phospholipase. In addition, bradykinin selectively stimulated prostaglandin E 9-ketoreductase activity from veins but not arteries. This may explain the finding that bradykinin induces the release of prostaglandin E compounds from arteries but prostaglandin F compounds from veins.     

Isolation of proteins with carbonyl reductase activity and prostaglandin-9-ketoreductase activity from chicken kidney

Kidney has the greatest capacity among the tissues of chicken for reducing aromatic ketones, and two ketone reductases were separated from this tissue by DEAE-cellulose chromatography and isolated. Though both are monomeric proteins with a molecular weight of 29,500, and with similar amino acid compositions and immunological properties, they differ in their pI values. The two enzyme species show no apparent difference in catalytic properties; aromatic ketones, aldehydes and quinones are reduced at high rates and alicyclic ketones such as 3-ketosteroids and prostaglandin E2 at low rates. The substrate affinity for several representative substrates at pH 7.2 is higher than that at the optimal pH of 6.3. Both enzymes prefer NADPH to NADH as a cofactor. Low NADP+-dependent reverse reactions occur with 9- and 15-hydroxyprostaglandins and certain alcohols as substrates. The enzymes show similar sensitivities to heavy metal ions, SH-reagents, quercitrin, indomethacin, and FMN.     

Purification and regulatory properties of chicken heart prostaglandin E 9-ketoreductase.

Prostaglandin E 9-ketoreductase was purified from chicken heart by ammonium sulfate fractionation, and DEAE-Sephadex, hydroxylapatite and phosphocellulose chromatography. Two peaks of activity were resolved during the phosphocellulose chromatographic step. Both peaks were stimulated by a substance that was not bound to the phosphocellulose column. This stimulatory substance was destroyed by treatment with phosphodiesterase and 0.1 M NaOH. It was heat-stable (100 degrees, 2 min), nondialyzable, and resistant to treatment with pronase, ribonuclease, and deoxyribonuclease; but it was dialyzable after heating or digestion with pronase. Sodium pyrophosphate also enhanced the activities of the prostaglandin E 9-ketoreductases as did angiotensin I; but not angiotensin II. In the presence of 3':5'-cyclic AMP, AMP, or several other ribonucleotides, the enhancing effects of the natural stimulatory substance, sodium pyrophosphate or angiotensin I were blocked, but these ribonucleotides themselves had little effect on the enzymes activity. The substrate specificities of the two prostaglandin E 9-ketoreductases were also studied. Both the 9-keto group and the 15-keto group of 15-ketoprostaglandin F2 alpha could be converted to the corresponding hydroxyl group; the 15-keto group was reduced faster than the 9-keto group. Prostaglandin D2, a prostaglandin with a 9-hydroxyl and an 11-keto group, could not be converted to prostaglandin F2 alpha nor could cyclohexanone be converted to cyclohexanol by the prostaglandin E 9-ketoreductase.     

Selective stimulation of venous prostaglandin E 9-ketoreductase by bradykinin.

The effects of bradykin on prostaglandin metabolism in canine mesenteric vessels were examined. Bradykinin stimulated microsomal prostaglandin synthesis in both artery and vein; this stimulation was more pronounced when [14C] hosphatidylcholine rather than [14C] arachidonate was used as the substrate for prostaglandin synthetase. This suggested that bradykinin enhanced a membrane phospholipase. In addition, bradykinin selectively stimulated prostaglandin E 9-ketoreductase activity from veins but not arteries. This may explain the finding that bradykinin induces the release of prostaglandin E compounds from arteries but prostaglandin F compounds from veins.     

The effect of i-mu-ts'ao on a partially purified prostaglandin E 9-ketoreductase from swine kidney

The enzyme system, prostaglandin E 9-ketoreductase, which catalyzes the reduction of prostaglandin E to form prostaglandin F has been partially purified from swine kidney. This NADPH-linked enzyme is studied spectrophotometrically. The KM of this enzyme for prostaglandin E2 was found to be 180 microM. Studies with the partially purified enzyme indicate that prostaglandin E 9-ketoreductase is affected by a Chinese herbal medicine i-mu-ts'ao (leonurus heterophyllus sweet). An increase in the concentration of i-mu-ts'ao aqueous extract may influence the conversion of prostaglandin E2 into prostaglandin F2 alpha. This finding offers a possible explanation for the physiological role of i-mu-ts'ao when it is treated as a medicine.     

Purification of prostaglandin E2-9-oxoreductase from human decidua vera

Prostaglandin E2-9- oxoreductase (PGE2-9-OR), the enzyme which converts prostaglandin E2 (PGE2) to prostaglandin F2 alpha (PGF2 alpha), has been detected in human decidua vera. A 105-fold purification was achieved when the centrifuged homogenate was fractionated sequentially by DEAE-Trisacryl, hydroxyapatite-agarose gel, ultrogel AcA 44 and Matrex gel blue A gel chromatographies. The following kinetic constants for PGE2-9-OR have been obtained. The equilibrium constant with respect to PGE2 is 83 microM, the Michaelis constant, Km, for PGE2 is 80 microM, for NADPH 1.6 microM. The maximal velocity for the forward reaction is V1 = .203 pmol/min. The enzyme was inhibited by progesterone, oestradiol-17 beta, cortisol and pharmaceutical drugs. An activating effect could be demonstrated with Ca2+ and oxytocin. The occurrence of PGE2-9-OR in the decidua vera suggests that this enzyme may be responsible for the transformation of PGE2 to PGF2 alpha in these tissues. This may be an important mechanism for the initiation and maintenance of uterine contractions.     

Nitric oxide (NO) inhibits prostaglandin E2 9-ketoreductase (9-KPR) activity in human fetal membranes

Nitric oxide (NO) synthesized by fetal membranes may act either directly inhibiting myometrium contractility or indirectly interacting with tocolytic agents as prostaglandins (PGs). Here we examined if NO could modulate prostaglandin E(2) 9-ketoreductase (9-KPR) activity in human fetal membranes (HFM). 9-KPR is the enzyme that converts PGE(2) into PGF(2alpha), the main PGs known to induce uterine contractility at term. Chorioamnion explants obtained from elective caesareans were incubated with aminoguanidine (AG), an iNOS inhibitor, or NOC-18, a NO donor. NOC-18 (2mM) increased PGE(2) production and diminished PGF(2alpha) synthesis in HFM. AG presented the opposite effect. When we evaluated the activity of 9-KPR by the conversion of [(3)H]-PGE(2) into [(3)H]-PGF(2alpha) and 13,14-dihidro-15-keto prostaglandin F(2alpha) (the PGF(2alpha) metabolite), we found that NOC-18 inhibited 9-KPR activity. Interestingly, AG did not elicit any effect on 9-KPR but l-NAME, a non-selective NOS inhibitor, significantly increased its activity. Our data suggests that exogenous NO inhibits 9-KPR activity in HFM, thus modulating the synthesis of important labor mediators as PGF(2alpha).     

Glutathione-prostaglandin A1 conjugate as substrate in the purification of prostaglandin 9-ketoreductase from rabbit kidney

Using GSH-PGA₁ as substrate for determination of enzyme activity a pI 4.8 form of rabbit kidney prostaglandin 9-keto-reductase has been purified 95 times to a specific activity of 1755 nmol/min per mg protein. The purification procedures involve ion-exchange chromatography, gel-filtration and affinity chromatography. The latter procedure comprises Blue Sepharose affinity chromatography, and GSH-PGA₁-Sepharose affinity chromatography.The purified enzyme preparation also showed a weak NADP⁺-dependent 15-hydroxyprostaglandin dehydrogenase activity, 20 nmol/min per mg protein with PGE₁ as substrate. K_m(PGE₁) for the dehydrogenase is 142.6 ± 45.1 μM (S.E., n=7).     

Metabolism of prostaglandin E1 and of glutathione conjugate of prostaglandin A1 (GSH-prostaglandin A1) by prostaglandin 9-ketoreductase from rabbit kidney.

Rabbit kidney prostaglandin 9-ketoreductase was found to metabolize the glutathione conjugate of prostaglandin A1 (GSH-prostaglandin A1). Apparent Km (GSH-prostaglandin A1) 13 microM and apparent Km (prostaglandin E1) 200 microM. The cytosolic preparation was subjected to gelfiltration and isoelectric focusing, which revealed that metabolism of prostaglandin E1 and GSH-prostaglandin A1 occurs by means of the same fractions. Furthermore, prostaglandin E1 and GSH-prostaglandin A1 are competitive inhibitors of the enzyme, when GSH-prostaglandin A1 and prostaglandin E1 are tested as substrates, respectively. It si concluded, that GSH-prostaglandin A1 is a much better substrate for prostaglandin 9-ketoreductase from rabbit kidney than is prostaglandin E1.     

15-Keto-13,14-dihydroprostaglandin E2- and F2 alpha-metabolite levels in blood from men and women given prostaglandin E2 orally

Prostaglandin E2 (PGE2) was administered orally in a dose of 1 mg to healthy males (n = 20) and females (n = 10). Blood levels of 15-keto-13,14-dihydroprostaglandin F2 alpha (PGF2 alpha-M) and 15-keto-13,14-dihydroprostaglandin E2 (PGE2-M), determined as the rearrangement product 11-deoxy-15-keto-13,14-dihydro-11 beta, 16-cycloprostaglandin E2 (PGE2-cyclo-M), were measured. The levels of the two PG metabolites increased already 10 minutes after ingestion of the tablet and the mean peak value for PGE2-cyclo-M in the men was 4.64 nmol/l which was reached 50 minutes after PGE2 administration. The mean peak value in women was 4.99 nmol/l which was obtained after 30 minutes. The increase in PGE2-cyclo-M concentration was significantly faster (p less than 0.05) in women than in the men. The mean plasma concentration of PGF2 alpha in males were 0.20 nmol/l prior to treatment and rose after PGE2 ingestion to mean peak level of 0.84 nmol/l after 70 minutes. The corresponding values for the females were 0.18 nmol/l and 0.88 nmol/l 50 minutes into treatment. When the data from both sexes were amalgamated PGE2-cyclo-M peak levels were reached significantly (p = 0.004) sooner than the PGF2 alpha-M peak. The two PG metabolites returned to baseline levels in 70% of the individuals after 240 minutes. The increase in PGF2 alpha-M concentration following oral administration of PGE2 indicates that part of the PGE2 was reduced to PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.