Purification and partial characterization of an NADH-linked delta13-15-ketoprostaglandin reductase from human placenta.

A Δ¹³-15-ketoprostaglandin reductase has been isolated from human placenta and purified 800-fold. The enzyme utilizes NADH as a cofactor but not NADPH. It reduces the 13,14 double bond in 15-ketoprostaglandin E₁, E₂ and F_2α. The K_M apparent for NADH is 54.8 μM and the K_M apparent for 15-ketoprostaglanding E₂ is 7.0 μM. The partially purified enzyme contains no 15-hydroxyprostaglandin dehydrogenase activity.     

Substrate specificity of three prostaglandin dehydrogenases

Studies on the substrate specificity, kcat/Km, and effect of inhibitors on the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase (9-ketoprostaglandin reductase) indicate that it is very similar to a human brain carbonyl reductase which also possesses 9-ketoprostaglandin reductase activity. These observations led to a comparison of three apparently homogeneous 15-hydroxyprostaglandin dehydrogenases with varying amounts of 9-ketoprostaglandin reductase activity: an NAD- and an NADP-linked enzyme from human placenta and an NADP-linked enzyme from rabbit kidney. All three enzymes are carbonyl reductases for certain non-prostaglandin compounds. The placental NAD-linked enzyme, which has no 9-ketoprostaglandin reductase activity, is the most specific of the three. Although it has carbonyl reductase activity, a comparison of the Km and kcat/Km for prostaglandin and non-prostaglandin substrates of this enzyme suggests that its most likely function is as a 15-hydroxyprostaglandin dehydrogenase. The results of similar comparisons imply that the other two enzymes may function as less specific carbonyl reductases.     

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.     

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).     

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.     

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.     

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.     

Adenine nucleotide metabolism of blood platelets. VIII. Transport of adenine into human platelets

Adenine uptake into human blood platelets is a carrier-mediated process with a K_m of 159±21 nM and a V of 100±10 pmoles/min per 10⁹ platelets (in citrated platelet-rich plasma). The Q₁₀ was 2.53±0.22. A pH optimum was found at 7.5. Washing of the platelets increased the K_m to 453±33 nM and V to 397±38 pmoles/min per 10⁹ platelets. The change in shape induced in platelets by ADP was accompanied by an increase in V (2 times) and K_m (1.5 times).Guanine (K_i 50 μM), hypoxanthine (K_i 390 μM), adenine-N′-oxide (K_i 40 μM), adenosine (K_i 100 μM), RA 233 (K_i 75 μM) and papaverine (K_i 15 μM) acted as competitive inhibitors. Adenosine at low concentrations, and prostaglandin E₁ gave inhibition at only high adenine levels. A similar inhibition was obtained with 2-deoxy-d-glucose. Sulfhydryl-group inhibitors, pyrimidines and ouabain had no effect.     

Inhibition by gossypol of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis

Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca²⁺-dependent protein kinase from pig testis. Gossypol inhibited Ca²⁺-dependent activity of the enzyme without affecting its basal activity. The IC₅₀ value (concentration causing 50% inhibition) was 31 μM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP (K_i = 31 μM) or lysine-rich histone (K_i = 30μM), and competitively with respect to phosphatidylserine (K_i = 2.1 μM). With Ca²⁺, irrespective of the presence or absence of 1,3-diolein, the compound lowered V_max and increased the apparent K_a for Ca²⁺. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrate in the testis for the enzyme located in nucleosome), with an IC₅₀ value of 88 μM. These results suggested that a phospholipid-sensitive Ca²⁺-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis.     

9-Hydroxyprostaglandin dehydrogenase in rat kidney cortex converts prostaglandin I2 into 15-keto-13,14-dihydro 6-ketoprostaglandin E1

15-Keto-13,14-dihydro 6-ketoprostaglandin E1 was positively identified by gas chromatography-mass spectrometry with negative-ion chemical ionisation detection from samples of rat kidney high-speed supernatant incubated with prostaglandin I2 in the presence of NAD+. A decreased formation of this product was observed when NAD+ was substituted with NADP+ and none was observed in the absence of nucleotide or substrate prostaglandin I2. Experiments with [9 beta-3H]prostaglandin I2 showed a time- and concentration-dependent loss of tritium which appeared as tritiated water, typical of reaction of [9 beta-3H]prostaglandin substrates with the enzyme, 9-hydroxyprostaglandin dehydrogenase. Time-course measurements of the appearance of tritiated water showed similar rates with 6-keto[9 beta-3H]prostaglandin F1 alpha and 15-keto-13,14-dihydro 6-keto[9 beta-3H]prostaglandin F1 alpha as substrates. These experiments suggest that the transformation of prostaglandin I2 and 6-ketoprostaglandin F1 alpha into the 15-keto-13,14-dihydro 6-ketoprostaglandin E1 catabolite occurs in this in vitro preparation via the corresponding 15-keto-13,14-dihydro catabolite of 6-ketoprostaglandin F1 alpha.     

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.     

Inhibition by indomethacin and aspirin of 15-hydroxy-prostaglandin dehydrogenase in vitro

15-Hydroxyprostaglandin dehydrogenase from bovine lung was purified 7.4 times to a specific activity of 1.4 mU/mg of protein. The isoelectric point was estimated to 5.4 and the molecular weight by gelfiltration to 40,000. K_m for prostaglandin E₁ and for NAD⁺ were found to be 3.4 μM and 1.1 × 10^?4M respectively. The enzyme was inhibited by indomethacin and aspirin. The indomethacin inhibition was found to be non-competitive to prostaglandin E₁ having a K_i=1.4 × 10^?4M and a K_i^′=1.6 × 10^?5M.     

The use of tritiated prostaglandins in metabolism studies. II: Kinetic isotope effect: an useful tool to investigate catabolizing sequence of prostaglandins

It is well established that prostaglandin catabolism involves sequential actions of a 15-hydroxyprostaglandin dehydrogenase, a 15-keto-prostaglandin delta 13-reductase and a 15-ketoprostaglandin reductase. This pathway must be confirmed in never investigated tissues before any enzyme assay is carried out. We have developed a new, simple, rapid and reliable method to investigate catabolizing sequence of prostaglandins based on the tritium kinetic isotope effect which occurs during the oxidation of the 15-hydroxyl group of the prostaglandin into a 15-keto group.     

Inhibition of 15-hydroxyprostaglandin dehydrogenase by papaverine.

Papaverine was found to inhibit NAD⁺-linked 15-hydroxyprostaglandin dehydrogenase partially purified from guinea pig lung. The inhibition was noncompetitive with prostaglandin E₂, uncompetitive with NAD⁺, and reversible. The Ki was calculated to be 26 μM. Papaverine also inhibited the enzyme from swine lung, chicken and dog heart, and rat and dog kidney. The inhibitory effects of papaverine on the 15-hydroxyprostaglandin dehydrogenase were compared with those on cyclic AMP phosphodiesterases in these tissues.     

The use of tritiated prostaglandins in metabolism studies II: Kinetic isotopic effect: An useful tool to investigate catabolizing sequence of prostaglandins

Although numerous data exist concerning tritium kinetic isotope effect in enzymic reactions, little is related to the metabolism of tritiated prostaglandins. The present study reports an evaluation of the kinetic isotope effect which occurs during the oxidation of 15-hydroxyl group of tritium-labeled prostaglandins E₂ and F_2α by the 15-hydroxyprostaglandin dehydrogenase and during the oxidation of 9-hydroxyl group of tritium-labeled prostaglandin F_2α by the 9-hydroxyprostaglandin dehydrogenase. The large kinetic isotope effect tends to limit the validity of the dehydrogenase assay using tritium-labeled prostaglandins as substrate. However these assays can be considered to be an indication of relative enzyme activity.     

Partial purification of the NADPH-dependent aldehyde reductase from bovine cardiac muscle.

An aldehyde reductase catalyzing the NADPH-dependent reduction of long-chain aldehydes has been purified 690-fold from bovine cardiac muscle. Based on the results obtained during gel filtration, this enzyme has an apparent molecular weight of 34,000. The pI of the aldehyde reductase was 6.1 and the enzymatic activity had a sharp pH optimum at 6.4. The enzyme catalyzed the reduction of aromatic aldehydes and aliphatic aldehydes having eight or more carbon atoms. Short-chain aldehydes, aldoses, or ketoses or long-chain methyl ketones were not utilized as substrates by this enzyme. However, the methyl ketone, pentadecan-2-one, was a competitive inhibitor of this enzyme with an apparent K_i = 10 μm when tetradecanal was the variable substrate. The reaction was not reversible when ethanol or hexadecanol was employed as substrate, utilizing either NAD⁺, or NADP⁺ as a cofactor. The addition of 10 mm pyrazole to the incubation medium had no effect on the enzymatic activity.     

Purification and properties of 6-phosphogluconate dehydrogenase fromMytilus galloprovincialis digestive gland

1. The enzyme 6-phosphogluconate dehydrogenase from digestive gland ofMytilus galloprovincialis was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis.2. The enzyme was purified 229-fold with a final specific activity of 2.3 μmol of NADP⁺ reduced/min per mg of protein and overall yield of 10%.3. The molecular weight of the native enzyme is estimated to be 100, 000 from gel-filtration studies.4. The influence of pH and MgCl₂ concentration on enzyme activity have been studied and the results have been compared to those reported by other authors for enzymes from different sources.5. TheK_mvalues for 6-phosphogluconate and NADP⁺ at room temperature (20°C) are approx. 20 and 40 μM respectively.6. NADPH was an inhibitor strictly competitive with respect to NADP⁺ (K_i = 14 μM) and non-competitive with respect to 6-phosphogluconate (K_i = 45 μM).     

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.     

The function of nickel on the urease activity of the lichen Evernia prunastri

The lichen Evernia prunastri synthesizes urease (EC 3.5.1.5) as a response to urea supplied in the medium. Nickel is required to produce the active protein. In addition, nickel behaves as an activator of the enzyme (K_a = 8.1 μM) at low concentrations whereas it is shown to be a competitive inhibitor of urease when it is supplied at concentrations higher than 40 μM (K_i = 50 μM).