Properties of solubilized prostaglandin synthetase from sheep vesicular glands.

Prostaglandin synthetase activity associated with the microsomal fraction from sheep vesicular glands has been solubilized by treatment with the non-ionic detergents Tween 20, Lubrol Px and Lubrol Wx. Approx.8 fold purification from microsomes is obtained and over 90% of the activity is recovered in the detergent solubilized fraction. The solubilized synthetase activity is stable at pH 5.0 but is gradually lost at pH 8.0; it is also heat and acid labile. The relative amounts of prostaglandins E₂, D₂ and F_2α formed by the microsomal-bound synthetase and by the solubilized synthetase are similar. Also similar are the pH optima (7.9–8.5) of the two synthetase preparations. The solubilization process appears to yield a fully active enzymatic preparation which could be employed for further purification and characterization of the prostaglandin synthetase complex.     

Purification and characterisation of prostaglandin endoperoxide synthetase from sheep vesicular glands.

The membrane-bound prostaglandin endoperoxide synthetase was purified until homogeneity, starting from sheep vesicular glands. The enzyme was obtained as a complex with Tween-20, containing 0.69 mg detergent per mg protein. No residual phospholipid could be detected. Prostaglandin endoperoxide synthetase appeared to be a glycoprotein, containing mannose and N-acetyl-glucosamine. No haemin or metal atoms were present. A molecular weight of 126 000 was found for the apoprotein by ultracentrifugation in 0.1% Tween solutions. The polypeptide chain without carbohydrate had a molecular weight of 69 000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The pure enzyme displays both cyclooxygenase and peroxidase activity, thus converting arachidonic acid into prostaglandin H2. The isolated synthetase requires haemin, which possibly acts as an easily dissociable prosthetic group, and a suitable hydrogen donor to protect the enzyme from peroxide inactivation and which is consumed in stoichiometric amounts to reduce the intermediate hydroperoxy group.     

Observations on the substrate specificity of prostaglandin hydroxylases of monkey seminal vesicles and sheep vesicular glands

Prostaglandin (PG) 19-hydroxylase of monkey seminal vesicles metabolizes PGE1 and PGE2 to their 19-hydroxy metabolites, while PGE2 20-hydroxylase of ram vesicular glands metabolizes PGE2 to 20-hydroxy-PGE2. The purpose of the present study was determine whether PGF2 alpha is a substrate of these enzymes. Deuterated 20-hydroxy-PGF2 alpha was employed as an internal standard to study the hydroxylation of PGF2 alpha (0.2 mM) by microsomes of monkey (Macaca fascicularis) seminal vesicles in the presence of NADPH, and the biosynthesis was compared with the hydroxylation of PGE2 under identical conditions. 19-Hydroxy-PGF2 alpha was formed at a rate of 3.5% of the formation of 19-hydroxy-PGE2. Microsomes of ram vesicular glands also hydroxylated PGE2 more efficiently than PGF2 alpha, which was converted to both 20-hydroxy-PGF2 alpha and 19-hydroxy-PGF2 alpha at a combined rate of 5% of the biosynthesis of 20-hydroxy-PGE2 under the same conditions. 20-Hydroxy-PGF2 alpha was demonstrated in ram semen, but the concentration was low (0.1 microM) in comparison with 20-hydroxy-PGE2 (24 microM). The two genital PG hydroxylases thus metabolize PGF2 alpha much less efficiently than PGE2. This finding may suggest that 19-hydroxy- and 20-hydroxy-PGF2 alpha in seminal fluids also could be formed by other mechanisms, e.g., from 19-hydroxy- and 20-hydroxy-PGE2 by the PGE 9-keto reductase enzyme.     

Structural characteristics of prostaglandin synthetase from sheep vesicular gland

Prostaglandin synthetase contains both oxygenase and peroxidase activity and catalyzes the first step of prostaglandin synthesis. Aspirin (acetylsalicylic acid) inhibits oxygenase activity by acetylating a serine residue of the enzyme. In the current study, we have investigated the subunit structure of this complex enzyme and the stoichiometry of aspirin-mediated acetylation of the enzyme. The enzyme was purified to near homogeneity in both active and aspirin-acetylated forms. The purified protein was analyzed for enzymatic activity, [3H]acetate content following treatment with [acetyl-3H]aspirin, NH2-terminal sequence, and amino acid composition. The results show first, that the enzyme can be purified to near homogeneity in an active form; second, that the enzyme consists of a single polypeptide chain (molecular weight 72,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis) with a unique NH2-terminal sequence (Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-Met-Gly-); and third, that aspirin inhibits the enzyme by transfer of one acetate per enzyme monomer. Therefore, the two distinct enzymatic activities, oxygenation and peroxidation, are present in a single polypeptide chain. Experiments with a cross-linking agent indicate that in nonionic detergent the enzyme is a dimer of two identical subunits.     

Effect of low and high methional concentrations on prostaglandin biosynthesis in microsomes from bovine and sheep vesicular glands

The effect of methional on prostaglandin biosynthesis from 5,8,11, 14-eicosatetraenoic acid was studied with microsomes from both bovine vesicular glands (BVG) and sheep vesicular glands (SVG). Ethylene was identified when methional was added to the fatty acid-microsome incubation systems showing that oxygen centered radicals such as hydroxyl radical were generated during incubation. A low methional level, 1 mM, enhanced the rate of prostaglandin biosynthesis in both BVG and SVG. A high methional level, 10 mM, inhibited prostaglandin biosynthesis in both BVG alone and SVG solubilized with 1% Tween 20. The inhibitory effect of 10 mM methional was reversed by lyophilization. These data suggest that oxygen centered radicals are used in prostaglandin biosynthesis even though they inactivate the enzyme complex.     

Effect of low and high methional concentrations on prostaglandin biosynthesis in microsomes from bovine and sheep vesicular glands.

The effect of methional on prostaglandin biosynthesis from 5,8,11,14-eicosatetraenoic acid was studied with microsomes from both bovine vesicular glands (BVG) and sheep vesicular glands (SVG). Ethylene was identified when methional was added to the fatty acid-microsome incubation systems showing that oxygen centered radicals such as hydroxyl radical were generated during incubation. A low methional level, 1 mM, enhanced the rate of prostaglandin biosynthesis in both BVG and SVG. A high methional level, 10 mM, inhibited prostaglandin biosynthesis in both BVG alone and SVG solubilized with 1% Tween 20. The inhibitory effect of 10 mM methional was reversed by lyophilization. These data suggest that oxygen centered radicals are used in prostaglandin biosynthesis even though they inactivate the enzyme complex.     

Characterization of gastric mucosal prostaglandin E2 receptor

1. The binding characteristics of gastric mucosal prostaglandin (PG) E2 (PGE2) receptor were investigated using mucosal cell membranes from rat stomach. The binding was found to be dependent upon PGE2 and membrane protein concentration, the time of incubation and the pH of the mixture, being highest at pH 3.0. 2. Scatchard analysis of the binding data revealed a curvilinear plot with high affinity binding (Kd = 2 nM; Bmax = 0.106 pmol/mg protein) and low affinity binding (Kd = 319 nM; Bmax = 2.262 pmol/mg protein) sites. 3. Competitive displacement study indicated that the receptor was specific for PGs of the E series, as PGF2 alpha and 6-keto-PGF1 alpha failed to displace the PGE2. 4. The study is the first report to provide biochemical parameters of specific PGE receptors in rat gastric mucosa.     

Immunochemical and kinetic evidence for two different prostaglandin H-prostaglandin E isomerases in sheep vesicular gland microsomes

Splenic lymphocytes from mice immunized with a partially purified prostaglandin (PG) H-PGE isomerase from sheep vesicular glands were fused with SP2/0-Ag14 myeloma cells. Two spleen cell-myeloma hybrids (hei-7 and hei-26) were selected and cloned. The mouse antibodies secreted by the two hybrids, IgG1 (hei-7) and IgG1 (hei-26), caused immunoprecipitation of a maximum of 45 and 22%, respectively, of the solubilized PGH-PGE isomerase activity of sheep vesicular gland; immunoprecipitation of activity by the two antibodies was additive. The antigens reactive with IgG1 (hei-7) and IgG1 (hei-26) were identified as proteins with Mr = 17,500 and 180,000, respectively, by Western transfer blotting or sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated 125I-labeled microsomes. The PGH-PGE isomerase activities precipitated by IgG1 (hei-7) and IgG1 (hei-26) exhibited different kinetic properties with respect to time course, Km for PGH2, and concentration dependence for GSH. No significant GSH-S-transferase activity was present in these immunoprecipitates. These data indicate that there are at least two different proteins in sheep vesicular gland microsomes capable of catalyzing GSH-dependent PGH-PGE isomerase reactions. IgG1 (hei-7), but not IgG1 (hei-26), caused coprecipitation of PGH synthase and PGH-PGE isomerase activities when incubated with intact right-side-out vesicular gland microsomes. Thus, the epitope for IgG1 (hei-7) is located on the cytoplasmic surface of those microsomal spheres which contain PGH synthase. This latter finding suggests that the isomerase reactive with IgG1 (hei-7) is involved in PGE synthesis in sheep vesicular glands.     

The heme-binding properties of prostaglandin synthetase from sheep vesicular gland

Purified, apoprostaglandin synthetase was prepared from sheep vesicular gland and studied in terms of its heme-binding properties. The enzyme binds a single heme group per enzyme monomer, Mr = 70,000. When reconstituted with heme, the enzyme has an absorption maximum at 412 nm and an absorption coefficient, epsilon 412 nm, of 120 mM-1 cm-1. The binding of heme to the apoenzyme was accompanied by a proportional increase in enzyme activity up to the point of heme-binding saturation. This reconstituted holoenzyme forms prostaglandin H2 from arachidonate. We conclude that prostaglandin synthetase possesses the heme-binding properties of a "typical" heme protein and that a single heme group mediates both the oxygenase and the peroxidase activities of the enzyme.     

Regulation of breathing movements in fetal sheep by prostaglandin E2

In fetal sheep, plasma prostaglandin (PG) E2 concentrations are high, and fetal breathing movements (FBM) occur intermittently, primarily during low-voltage fast electrocortical activity (LVFA). There is evidence suggesting that prostaglandins, specifically PGE2, may regulate FBM. To define the physiological role of PGE2 in regulation of FBM, we infused meclofenamate (0.9 mg X kg-1 X h-1), a prostaglandin synthetase inhibitor, into six fetal sheep to suppress endogenous prostaglandin production. Afterward, PGE2 was added in mean doses of 9, 18, 36, and 90 ng X kg-1 X min-1. Meclofenamate decreased PGE2 concentrations and increased FBM, especially during high-voltage slow electrocortical activity (HVSA). Addition of PGE2 reversed the effects of meclofenamate, increasing PGE2 concentrations and decreasing FBM, especially during HVSA. The response to PGE2 was dose dependent; the overall incidence of FBM and incidences of FBM during HVSA and LVFA were inversely correlated with both the infused PGE2 dose and the mean PGE2 concentration. At higher doses of PGE2, FBM occurred intermittently and only during LVFA; thus PGE2 infusion restored the physiological pattern of FBM. These results indicate that PGE2 regulates FBM by inhibiting FBM during HVSA.     

The effect of haptoglobin on prostaglandin H synthase from ram vesicular glands

It was shown that the ability of sheep and horse haptoglobins differing in their immunological properties to inhibit PGH synthetase is about the same. It was found that haptoglobin inhibits the PGH synthetase-catalyzed enzymatic reaction, the inhibiting effect being non-competitive with respect to the electron donor, adrenaline. The degree of PGH synthetase inhibition by haptoglobin depends on the glycoprotein concentration, incubation time and enzyme activity.     

Identification of haptoglobin as an endogenous inhibitor of prostaglandin H synthase in the cytosol fraction of primary cells from sheep vesicular glands

It was shown that cytosol of primary sheep vesicular gland cells inhibits peroxidase activity of prostaglandin H synthase (PGHS). The degree of the enzyme inactivation depends on cytosol concentration. It was established that cytosol contains glycoprotein haptoglobin that is one of the cytosol basic components responsible for its property to inhibit PGHS. Haptoglobin is supposed to participate in endogenous regulation of PGHS activity in sheep vesicular glands.     

Inhibition of prostaglandin synthase activity of sheep seminal vesicular gland by human serum haptoglobin

Serum haptoglobin was added to the reaction mixture of prostaglandin synthase (EC 1.14.99.1) and its inhibitory effect was studied [1-14C]Arachidonic acid was used as substrate and the enzyme activity was estimated by monitoring the radioactivity of the products after thin layer chromatography. With or without addition of hemoglobin to the reaction mixture, both the purified haptoglobin 1-1 and 2-2 showed inhibitory activity. In the presence of 5 microM hematin, however, inhibitory activity haptoglobin was not observed. Inhibition of prostaglandin synthesis in the system depended on the molar ratio of haptoglobin to hemoglobin in the reaction mixture. These results demonstrate that haptoglobin inhibits prostaglandin synthase by restricting available heme group for the enzyme activity through complexing with hemoglobin. However, haptoglobin did not inhibit completely the stimulatory effect of free hemoglobin. Relevant significant of this effect was discussed.     

Characterization of opioid peptides from maternal and fetal sheep adrenal glands

Enkephalin immunoreactive material from adrenal glands was characterized both in maternal and fetal sheep at various gestational ages. Whole gland extracts from both maternal and fetal sheep contained three major peaks of Enk immunoreactivity corresponding to apparent molecular weights of 10,000, 2800, and less than 1200 daltons. The majority of maternal adrenal Enk immunoreactivity was found in medullary tissue, although cortex also contained low but detectable amounts. This was also the case in newborn lambs and 139 day fetuses, where adrenal cortex was sufficiently developed to allow extraction and quantitation of opioid material. In fetuses at mid-gestation (70-80 days), adrenal medullary Enk immunoreactivity was approximately 75% of maternal values. Met-Enk and Leu-Enk content in 139 day fetal medulla were 70 and 76% of maternal values respectively, while newborn Met- and Leu-Enk medullary content were similar to maternal values. The molar ratio of Met-Enk to Leu-Enk was approximately 4:1 in both maternal and fetal adrenal medulla, and 2:1 in adrenal cortex, suggesting different synthetic processing of opioid peptides in the two tissues. The early appearance of significant levels of adrenal medullary Enk immunoreactivity and subsequent development paralleling that of catecholamines suggest a predominant role for adrenal enkephalins in regulation of fetal cardiovascular function early in gestation.     

The relationship between fetal breathing movements and prostaglandin E2 during ACTH-induced labour in sheep

We measured fetal breathing movements and fetal carotid arterial prostaglandin E concentrations during adrenocorticotrophin-induced labour in 6 pregnant sheep and in 6 control animals starting at day 127. The 6 ACTH-treated animals went into labour on average 97 h after the onset of infusion and the incidence of fetal breathing movements diminished during the last 12h before the onset of labour. There was a significant negative relationship between the incidence of fetal breathing movements and fetal carotid arterial prostaglandin E concentrations (r = -0.88; P less than 0.001) in ACTH treated animals. These data suggest a role for prostaglandin E in the diminution of fetal breathing movements prior to the onset of labour.