Isolation of two proteins with 9-ketoprostaglandin reductase and NADP-linked 15-hydroxyprostaglandin dehydrogenase activities and studies on their inhibition

Two proteins containing 9-ketoprostaglandin reductase and NADP-linked 15-hydroxyprostaglandin dehydrogenase activities have been isolated. Both of these activities are inhibited by a low molecular weight placental protein and by indomethacin, ethacrynic acid, and furosemide.     

A simple and sensitive assay for 9-hydroxyprostaglandin dehydrogenase

The tritium recovery assay of 9-hydroxyprostaglandin dehydrogenase [Pace-Asciak, C. (1975) J. Biol. Chem.250, 2789] has been modified to ensure its applicability to both crude and purified enzyme preparations. The stereospecificity of NAD⁺-dependent 9-hydroxyprostaglandin dehydrogenase with respect to NAD⁺ was determined first and found to be A-side specific. Based on the stereospecificity of the enzyme, a simple and sensitive assay method for 9-hydroxyprostaglandin dehydrogenase has been developed. The assay is able to detect picomole quantities of substrate conversion. When 15-keto-13,14-dihydro-[9β-³H]PGF_2α is employed as substrate, the tritium label of the tritiated prostaglandin is effected to transfer to lactate stereospecifically by coupling 9-hydroxyprostaglandin dehydrogenase with a saturating level of lactate dehydrogenase. The amount of prostaglandin oxidized is quantitated by the radioactivity of the labeled lactate produced, which is separated from labeled prostaglandin by charcoal precipitation. Simultaneous assays with the current tritium-release and thin-layer chromatography methods indicated excellent correlation. Using this method we have found that rat kidney possesses the highest enzyme activity among those tissues examined. Rat kidney enzyme activity is linear for the first 10 min it is studied and is nonlinear with increasing amounts of crude enzyme extract, indicating the possible presence of endogenous inhibitor(s). The apparent K_m for 15-keto-13,14-dihydro-PGF_2α is 0.66 μm. The enzyme is activated by imipramine, inhibited by indomethacin, but not affected by furosemide and ethacrynic acid. These results confirm previous findings reported in the literature.     

NADP-linked 15-hydroxyprostaglandin dehydrogenase from human placenta: partial purification and characterization of the enzyme and identification of an inhibitor in placental tissue.

An NADP-linked 15-hydroxyprostaglandin dehydrogenase has been identified in human placental tissue and partially purified. Prostaglandins of the A and B series are good substrates for this enzyme while those of the E and F series are not. This enzymic preparation also catalyzes oxido-reductions at the 9 position of the prostaglandin molecule; these are slow compared to those occurring at the 15 position of the prostaglandins in the A and B series. Disc gel electrophoresis of the purified enzyme reveals the presence of three protein bands which contain dehydrogenase activity. Boiled placental homogenates contain an inhibitor which appears to be specific for the NADP-linked 15-hydroxyprostaglandin dehydrogenase. The inhibitor is heat stable and has a molecular weight of 6,000 – 7,000.     

An NADP-linked prostacyclin dehydrogenase in rabbit kidney

An NADP-linked 15-hydroxyprostaglandin dehydrogenase specific for prostacyclin was purified 1,300-fold from rabbit kidney. Prostaglandins E₂, F_2α, and 6-Keto PGF_1α and thromboxane B₂ were oxidized by the purified enzyme with rates of reaction less than 4% that of PGI₂. Unlike other rabbit kidney NADP-linked 15-hydroxyprostaglandin dehydrogenases, this enzyme catalyzes oxido reduction more rapidly at the 15- position than at the 9- position and does not utilise NAD as a cofactor. It has a molecular weight of 62,000 and migrates on polyacrylamide disc gel electrophoresis as a single diffuse band. The reaction product was identified by thin-layer chromatography as 6,15-diketo PGF_1α. Prostacyclin dhydrogenase is the first 15-hydroxyprostaglandin dehydrogenase described which is specific for the metabolism of prostacyclin.     

A ubiquitin-like peptide from the mushroom Pleurotus sajor-caju exhibits relatively potent translation-inhibitory and ribonuclease activities

The fruiting bodies of the edible mushroom Pleurotus sajor-caju were extracted with an aqueous buffer and then subjected to affinity chromatography on Affi-gel Blue gel, ion exchange chromatography on DEAE-cellulose and gel filtration on Superdex 75. From the fraction of the extract adsorbed on Affi-gel Blue gel and unadsorbed on DEAE-cellulose, a 9.5 kDa peptide with an N-terminal sequence similar to ubiquitin was isolated with a yield of 0.25 mg/kg mushroom. The peptide inhibited cell-free translation with an IC(50) of 30 nM. It exhibited a ribonuclease activity of 450 U/mg toward yeast transfer RNA. The activities were substantially more potent than those of previously isolated mushroom ubiquitin-like protein and peptide.     

Kinetic studies on a 15-hydroxyprostaglandin dehydrogenase from human placenta.

Kinetic studies have shown that the reaction catalyzed by the human placental 15-hydroxyprostaglandin dehydrogenase proceeds by a single displacement mechanism. Addition of the reactants is ordered with NAD⁺ binding first. The lifetime of the ternary complex is affected by the pH of the reaction mixture. At pH 7.0 a kinetically significant ternary complex is formed, while at pH 9.0 the ternary complex is not kinetically significant (Theorell-Chance mechanism). There is evidence for the occurrence of a kinetically significant isomerization of the enzyme · NADH complex at pH 9.0 but not at pH 7.0. At high substrate concentrations there is formation of unreactive complexes between the 15-hydroxyrostaglandin and both the free enzyme and enzyme · NADH complex and between the 15-ketoprostaglandin and both the free enzyme and enzyme · NAD⁺ complex. The inhibition of the 15-hydroxyprostaglandin dehydrogenase by various prostaglandins and prostaglandin analogs may be explained by the formation of similar unreactive complexes. Certain prostaglandin analogs, arachidonic acid, and ethacrynic acid also affect the activity of the enzyme by causing its irreversible inactivation.     

Characterization of two enzyme proteins catalyzing NADP+/NADPH-dependent oxidoreduction of prostaglandins at C-9 and C-15 from swine kidney

Two proteins (pI 4.8 and 5.8) capable of catalyzing NADP⁺/NADPH-dependent oxidoreduction of prostaglandins at C-9 and C-15 but not at C-11 have been purified to homogeneity from swine kidney. Both proteins exhibited identical molecular weight and subunit size. Similar amino acid composition, antigenic determinants, and coenzyme and substrate specificity were also found. The molecular weight of the enzyme as determined by gel filtration was 29,000. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gel gave a value of 29,500 indicating the presence of a single polypeptide chain. Either enzyme protein utilized a variety of prostaglandins (PGS) as substrates. PGA₁-glutathione conjugate and PGB₁ were found to be the best substrates for prostaglandin 9-ketoreductase and 15-hydroxyprostaglandin dehydrogenase activities, respectively. For prostaglandins having dual reactive groups in a single molecule, the rate of oxidation of PGF_2α at C-15 was comparable to that at C-9, whereas the rate of reduction of 15-keto-PGE₂ at C-15 was far greater than that at C-9.     

Cloning and sequence analysis of the cDNA for human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase catalyzes the oxidation of many prostaglandins at C-15, resulting in a subsequent reduction in their biological activity. We report the isolation of the cDNA for this enzyme. A human placental lambda gt11 cDNA library was screened using polyclonal antibodies prepared against the human placental enzyme. A 2.5-kilobase cDNA containing the entire coding region for the enzyme was isolated. The cDNA encodes for a protein of 266 amino acids with a calculated Mr of 28,975. Identification of the cDNA as that coding for 15-hydroxyprostaglandin dehydrogenase was based on the comparison of the deduced amino acid sequence with the amino acid sequence of two peptides, one from the rabbit lung enzyme and the other from the human placental enzyme. This cDNA hybridizes with two species of poly(A+) RNA isolated from human placenta: one of 3.4 kilobases and the other of 2.0 kilobases. Isolation of the cDNA for 15-hydroxyprostaglandin dehydrogenase should facilitate studies on the structure, function, and regulation of this enzyme.     

Purification and Properties of Neutral Proteinase I from Aspergillus oryzaePurification and Properties of Neutral Proteinase II from Aspergillus oryzae

Neutral proteinase I (the first peak in DEAE-cellulose chromatogrraphy) was purified from the Amberlite IRC-50 adsorbed fraction by chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. It shows an optimum pH of 7.0 for milk casein. The enzyme was found to be stable in the pH range of 5.5 to 12.0. The molecular weight of the enzyme was estimated to be about 41,000 by gel filtration. The enzyme had neither aminopeptidase nor carboxypeptidase activity, but degraded carbobenzoxy-glycyl-phenyl-alanine amide, poly-l-lysine and poly-l,α-glutamic acid. The enzyme was inhibited by ethylenediaminetetraacetate, but not inhibited by diisopropylphosphorofluoridate and potato inhibitor.     

Microspectrophotometric and enzymatic analyses of fish plasma proteins electrophoretically separated in thin polyacrylamide gels

A system for horizontal, discontinuous electrophoresis in thin sheets of polyacrylamide gel was developed to permit rapid and direct comparison of multiple samples of fish plasmas for population studies. The resulting electropherograms are suitable either for screening for enzyme polymorphisms or for densitometric scanning after gels are stained for total proteins. Each gel sheet (110×90×0.8 mm) can be used for simultaneous separation of 10–15 different samples. Since total voltage and the running time required for successful resolution of fine protein bands are greatly reduced in this system, minimal heating occurs within the gel matrix during electrophoresis. Up to 120 different blood samples (8 gel sheets) can be processed in about 8 h. Gel sheets are routinely stained overnight in Coomassie Brilliant Blue (0.1%, v/v), rinsed several times and stored overnight in 7% acetic acid to complete destaining. Particular gel sectors are then transferred to distilled water and mounted on glass microslides for photography or for densitometric evaluation with a Leitz microspectrophotometer. These procedures have been used to identify characteristic differences in the electrophoretic mobilities of plasma enzymes and albumin fractions from several populations of poeciliid fishes. Observed differences in albumin mobilities (albumin phenotypes) were verified by mixing isoaliquots of test plasmas with plasma samples containing albumins of known mobility. Resultant patterns for albumin bands for such mixed plasmas were indistinguishable from those obtained with plasma samples from the F₁ hybrid progeny of parents possessing albumins of characteristically different electrophoretic mobilities. Procedural details for gel casting, electrophoresis and sample evaluation are described.     

Identification of nitric oxide and nitrous oxide as products of nitrite reduction by Pseudomonas cytochrome oxidase (nitrate reductase)

The cytosol fraction of rat adrenocortical tissue contains comparatively high levels of two prostaglandin metabolizing enzymes. The first, prostaglandin-9-ketoreductase, utilizes NADPH more effectively than NADH as cofactor, is inhibited by NADP, and exhibits an apparent K_m of 304 μM for PGE₁. 15-hydroxyprostaglandin dehydrogenase, tentatively identified as the type II NADP-dependent isozyme, is inhibited by NADPH but not NADH, and exhibits an apparent K_m of 157 μM when PGE₁ is used as substrate. Changes in specific activities of the two enzymes following ACTH, hypophysectomy, or dexamethasone treatment are inconclusive in defining a chronic regulatory role for adrenocorticotropin.     

Phosphoprotein from Dentin. New Approaches to Achieve and Assess Purity

Phosphoprotein extracted from rat incisors was purified by passage through a sulfonated polystyrene column. The phosphoprotein that emerged in the void volume contained 54% phosphoserine + serine and 36% aspartic acid and, in contrast to that obtained by DEAE-cellulose chromatography, was devoid of proline, valine, isoleucine, leucine, tyrosine, phenylalanine and arginine. Gel electrophoresis of the material purified on sulfonated polystyrene columns gave one major phosphate-containing band which would not stain with Coomassie Blue. EDTA or acetic acid demineralization yielded phosphoprotein preparations with identical compositions and electrophoretic properties. These data show that purification procedures reported earlier are insufficient.     

Purification and properties of D-3-hydroxybutyrate dehydrogenase from Paracoccus denitrificans

D-3-Hydroxybutyrate dehydrogenase from Paracoccus denitrificans has been purified to near homogeneity. The enzyme was prepared using DEAE-cellulose chromatography, affinity chromatography on immobilized Cibacron blue (Matrex Gel Blue A) and gel permeation chromatography. The pure enzyme was obtained by chromatofocusing as the final isolation step. The purification procedure yielded the enzyme with a specific activity of about 100 units/mg protein. The enzyme is specific for D-3-hydroxybutyrate and NAD and it exhibits anomalous kinetics (hysteresis) at low enzyme and coenzyme concentrations. It is relatively stable in the presence of EDTA at pH 7–8 higer salt concentrations. D-3-Hydroxybutyrate dehydrogenase is a tetramer with a molecular weight of 130 000 ± 10 000, its isoelectric point equals 5.10 ± 0.05. The enzyme is applicable to the determination of acetoacetate and D-3-hydroxybutyrate concentrations.     

Characterization of Enterobacter cloacae and E. sakazakii by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases of 34 strains of Enterobacter cloacae and 22 strains of Enterobacter sakazakii were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. The two species could be separated on the basis of distinct electrophoretic patterns of all enzymes analysed. Glutamate dehydrogenase and acid phosphatase were detected exclusively in E. cloacae, whereas esterase bands were more intensively stained in E. sakazakii. For each species, two zymotypes could be distinguished, on the basis of electrophoretic mobilities of malate dehydrogenase and banding patterns of esterase for E. cloacae, and by both isoelectric point and electrophoretic mobilities of an esterase and of lactate and malate dehydrogenases for E. sakazakii. The high degree of enzyme polymorphism within the two species permitted precise identification of strains. The variations in electrophoretic patterns might therefore provide useful epidemiological markers.     

A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa

A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 μM. The protease did not have antifungal or ribonuclease activity.     

Spectrofluorometric assay of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity

A simple, rapid, and sensitive spectrofluorometric assay for 15-hydroxyprostaglandin dehydrogenase activity was developed in which the rate of production of NADH was monitored. The cytosolic fraction prepared from human placental tissue was employed as the enzyme source. The assay was conducted at pH 9.5 since 15-ketoprostaglandin Δ¹³-reductase and NADH oxidase activities were inhibited at this pH, thereby minimizing the interference of the reactions catalyzed by these enzymes in the assay of prostaglandin dehydrogenase activity.     

Glutathione-related inhibition of prostaglandin metabolism

Placental homogenates contain a heat-stable, dialyzable fraction which specifically inhibits two placental enzymes, both of which possess 15-hydroxyprostaglandin dehydrogenase and 9-ketoprostaglandin reductase activities. The inhibition of the two enzymes is the same. The inhibitor has been resolved into two components by gel filtration on a column of Sephadex LH-20. The component which eluted first has been identified as oxidized glutathione (GSSG), the other as a glutathione-containing material (GSX). Inhibition of the 15-hydroxyprostaglandin dehydrogenase activity is competitive with respect to the prostaglandin substrate (K_i^GSSG = 26 μM, K_i^GSX = 1.4 μM). Inhibition of the 9-ketoprostaglandin reductase activity is also competitive with respect to the prostaglandin substrate (K_i^GSSG = 68 μM). The most effective inhibitor of the 15-hydroxyprostaglandin dehydrogenase is the prostaglandin A₁-glutathione adduct (K_i = 0.27 μM). This compound is not a substrate for oxidation of the 15-hydroxyl group but it is the best substrate found to date for reduction of the 9-keto function.     

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.     

Hydrolysis of solubilized fibroin and silk proteins in the midgut of the pharate adult of Bombyx mori

Midgut protease in the pharate adult hydrolysed native silk proteins and solubilized fibroin by ethylenediamine cupric hydroxide or lithium bromide. By agar gel electrophoresis one to three protease bands moving toward the anode were detected, and the number of bands and the electrophoretic mobility were different among the various strains. Optimal activity of the enzyme was at about pH 8·3. The protease activity was found to decrease in higher concentrations of the substrates. One peak of protease activity was seen in Sepharose 6B chromatography, and the elution pattern and peak position of the enzyme were very similar to those of protease activity with casein. In DEAE-cellulose chromatography, the peak of activity for casein overlapped but did not coincide with a broad peak of protease hydrolysing native silk proteins. The results obtained support the assumption that the midgut protease in the pharate adult is one of the sources of the cocoon-digesting enzyme.     

Co-purification and characterization of ATP-sulfurylase and adenosine-5'-phosphosulfate kinase from rat chondrosarcoma

The two sulfate-activating enzymes, ATP-sulfurylase (EC 2.7.7.4) and adenosine-5'-phosphosulfate kinase (adenylylsulfate kinase, EC 2.7.1.25), were each purified about 2000-fold from crude rat chondrosarcoma homogenate. Throughout a purification protocol which included Sephacryl S-300 gel filtration, DEAE-Sephadex ion exchange, hydroxylapatite, and ATP-agarose affinity chromatography, these two activities consistently co-purified. ATP-sulfurylase and adenosine-5'-phosphosulfate kinase each showed a pH optima of 7.0-7.4 and a bimodal temperature optima of 46 and 52-54 degrees C. Both activities preferred Mg2+ as their divalent cation source over Mn2+, Co2+, or Zn2+. The apparent Km values determined for adenosine 5'-phosphosulfate in both assays was 1-5 microM; the Km for pyrophosphate in the sulfurylase reaction was 40 microM and for ATP in the kinase reaction was 5 mM. Gel electrophoresis indicated major bands at Mr = 160,000 in nondenaturing systems and 35,000-37,000 and 60,000 under dissociative conditions, whereas gel filtration of the most highly purified fractions yielded a coincident peak in the molecular weight range 260,000.