Rat striatal synaptosomes as a model system for studying the inhibition of dihydropteridine reductase activity

The distribution of dihydropteridine reductase between soluble and particulate fractions in synaptosomes parallels that of lactate dehydrogenase, but not monoamine oxidase. Ki and I50 values for inhibitors obtained with the enzyme-rich P2 fraction and its twice-washed fraction (P2W2) were essentially the same, and were similar to those obtained with highly purified human liver enzyme. Dihydropteridine reductase inhibitory potency of multi-ring compounds containing a catechol-moiety was greater than that of single ring catecholic compounds, which in turn was greater than that of p-hydroxy-phenolic compounds. The P2 fraction of rat striatal synaptosomal preparations may serve as a convenient source of dihydropteridine reductase for studying the inhibition of this enzyme.     

Partial purification and characterization of NADPH-linked aldehyde reductase from monkey brain

Abstract— The activity of NADPH-linked aldehyde reductase (EC 1.1.1.2) in various regions of monkey brain was determined in vitro. The highest specific activity of the enzyme was found in areas of the brain stem; including the pons, medulla and midbrain. A greater than 500-fold purification of the monkey brain enzyme was obtained by a combination of ammonium sulphate fractionation and subsequent chromatography on calcium phosphate gel cellulose and DEAE-cellulose. The aldehyde metabolites of the biogenic amines, norepinephrine, serotonin, dopamine and octopamine, were readily reduced by the NADPH-linked aldehyde reductase. The K_m values for 3,4-dihydroxyphenylglycolaldehyde, 3,4-dihydroxyphenyl-acetaldehyde, and 5-hydroxyindoleacetaldehyde were 12.0 μm , 6.1 μm and 27 μm , respectively. The maximum velocity (V_max) for 3,4-dihydroxyphenylglycolaldehyde was, respectively, five-fold or three-fold greater than that determined for 3,4-dihydroxyphenylacetaldehyde or 5-hydroxyindoleacetaldehyde. The highly purified enzyme derived from monkey brain was markedly inhibited by barbiturates, diphenylhydantoin, and chlorpromazine, but not by pyrazole. From data obtained by sucrose density gradient centrifugation and Sephadex chromatography the molecular weight of aldehyde reductase was determined to be about 70,000 daltons.     

Developmental changes in the distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase among subcellular fractions of rat brain.

—The distribution of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) relative to that of several biochemical markers has been studied in subcellular fractions prepared from the brains of rats, aged 4 days to adult, by differential centrifugation. In the brains of 10-day-old animals fractions which sedimented at 800 g (P₁ and 9000 g (P₂) contained 28% and 65% respectively of the total reductase activity. A similar distribulion of the microsomal marker, NADPH-cytochrome c reductase, suggested that the HMG-CoA reductase activity in the low-speed pellets was due to substantial contamination of these fractions with endoplasmic reticulum. When P₂ was fractionated on a discontinuous sucrose gradient, the distributions of protein, RNA and NADPH-cytochrome c reductase paralleled that of HMG-CoA reductase, indicaling a non-specific association of endoplasmic reliculum and HMG-CoA reductase with all of the structures sedimenting in P₂. As brain maturation proceeded and a greater percentage of total brain protein (primarily associated with myelin) sedimenled in P₁, the subcellular distributions of HMG-CoA reductase and the microsomal marker changed in a parallel way. By 21 days P₁ contained nearly all of the reductase activity. Because the specific activity of HMG-CoA reductase in P₁ decreased steadily between 4 and 21 days, while the specific activity of 2′:3′-cyclic nucleotide 3′-phosphohydrolase in this fraction increased in a coordinate fashion, we conclude that the reductase is not an integral component of myelin, and probably is associated exclusively with the endoplasmic reticulum included in P₁. In view of the developmental changes in the distribution of HMG-CoA reductase among subcellular fraclions, we suggest that whole homogenates (or comparable tissue extracts) should be utilized to evaluate reductase activity in the developing brain.     

Aldose reductase inhibitors from Litchi chinensis Sonn

Diabetes is one of the major risk factors for cataract. Aldose reductase has been reported to play an important role in sugar-induced cataract. In this study, we conducted pharmacological investigations upon experimental rat lenses using extracts of the fruits of Litchi chinensis (Sapindaceae). Of the extracts and organic fractions of L. chinensis tested, a MeOH extract and an EtOAc fraction were found to be potent inhibitors of rat lens aldose reductase (RLAR) in vitro — their IC₅₀ values being 3.6 and 0.3 μg/mL, respectively. From the active EtOAc fraction, four minor compounds with diverse structural moieties were isolated and identified as d-mannitol (1), 2,5-dihydroxybenzoic acid (2), delphinidin 3-O-β-galactopyranoside-39,59-di-O-β-glucopyranoside (3), and delphinidin 3-O-β- galactopyranoside-39-O-β-glucopyranoside (4). Among these, 4 was found to be the most potent RLAR inhibitor (IC₅₀ = 0.23 μg/mL), and may be useful in the prevention and/or treatment of diabetic complications.     

Purification and physicochemical properties of NADPH-specific dihydropteridine reductase from bovine and human livers

A new type of dihydropteridine reductase [EC 1.6.99.10], which is specific for NADPH as the substrate in the reduction of quinonoid-dihydropterin to tetrahydropterin, was purified to homogeneity from bovine liver and human liver. The molecular weight of the enzyme was determined to be 65,000-70,000. The enzyme was composed of two subunits with identical molecular weight of 35,000; the amino terminal residue was determined to be valine. The isoelectric point of the enzyme was 7.05. The physicochemical properties of this enzyme were quite different from those of bovine liver NADH-specific dihydropteridine reductase [EC 1.6.99.7]. NADPH-specific dihydropteridine reductase did not cross-react with an antiserum raised against the NADH-specific dihydropteridine reductase, nor did the latter enzyme react with an antiserum to the former enzyme, indicating that the two enzymes have no common antigenic determinants. NADPH-specific dihydropteridine reductase from human liver was shown to have properties similar to those of the bovine liver enzyme.     

Aldose reductase inhibition of a saponin-rich fraction and new furostanol saponin derivatives from Balanites aegyptiaca

BackgroundBalanites aegyptiaca Del. (Zygophyllaceae) fruits are used to treat hyperglycemia in Egyptian folk medicine and are sold by herbalists in the Egyptian open market for this purpose. Nevertheless, the fruits have not yet been incorporated into pharmaceutical dosage forms. The identity of the bioactive compounds and their possible mechanisms of action were not well understood until now.PurposeAldose reductase inhibitors are considered vital therapeutic and preventive agents to address complications caused by hyperglycemia. The present study was carried out to identify the primary compounds responsible for the aldose reductase inhibitory activity of Balanites aegyptiaca fruits.Study designThe 70% ethanolic extract of Balanites aegyptiaca fruit mesocarp and its fractions were screened for inhibition of the aldose reductase enzyme. Bio-guided fractionation of the active butanol fraction was performed and the primary compounds present in the saponin-rich fraction (D), which were responsible for the inhibitory activity, were characterized. HPLC chromatographic profiles were established for the different fractions, using the isolated compounds as biomarkers.MethodsAldose reductase inhibition was tested in vitro on rat liver homogenate. The butanol fraction of the 70% ethanolic extract was fractionated using vacuum liquid chromatography (VLC, RP-18 column). The most active sub-fraction D, which was eluted with 75% methanol, was subjected to preparative HPLC to isolate the bioactive compounds.ResultsThe butanol fraction displayed inhibitory activity against the aldose reductase enzyme (IC₅₀ = 55.0 ± 6 µg/ml). Sub-fraction D exhibited the highest inhibitory activity (IC₅₀ = 12.8 ± 1 µg/ml). Five new steroidal saponin derivatives were isolated from this fraction. The isolated compounds were identified as compound 1a/b, a 7:3 mixture of the 25R:25S epimers of 26-O-β-D-glucopyranosyl-furost-5-ene-3,22,26-triol 3-O-[α-L-rhamnopyranosyl-(1→3)- β-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside; compound 2, 26-O-β-D-glucopyranosyl-(25R)-furost-5-ene-3,22,26-triol 3-O-[ β-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside; compound 3, 26-O-β-D-glucopyranosyl-(25R)-furost-5,20-diene-3,26-diol 3-O-[α-L-rhamnopyranosyl-(1→3)- β-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside; compound 4, 26-O-β-D-glucopyranosyl-(25R)-furost-5,20-diene-3,26-diol 3-O-[ β-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside; and compound 5, which is the 25S epimer of compound 4, by using various spectroscopic methods [MS,1D and 2D NMR (HSQC, HMBC, DQF-COSY, HSQC-TOCSY)]. Compounds 1a/b, 2, 3, 4, 5 exhibited highly significant aldose reductase inhibitory activities (IC₅₀ values were 1.9 ± 0.2, 1.3 ± 0.5, 5.6 ± 0.2, 5.1 ± 0.4, 5.1 ± 0.6 µM, respectively) as compared to the activity of the reference standard quercetin (IC₅₀ = 6.6 ± 0.3 µM).ConclusionThe aldose reductase inhibitory activity of Balanites fruits is due to the steroidal saponins present. HPLC chromatographic profiles of the crude butanol fraction and its 4 sub-fractions showed that the most highly bioactive fraction D contained the highest amount of steroidal saponins (75%) as compared to the 21% present in the original butanol fraction. The isolated furostanol saponins proved to be highly active in an in vitro assay.     

A specific kinetic assay for phenylalanine hydroxylase

An assay procedure is given which is speedy, accurate, and specific, permitting direct recording of velocities, and obviating the use of reagents other than those necessary for the enzymatic reaction itself. The method is suitable for the study of enzyme mechanism and inhibition and also offers distinct advantages when used for other purposes, e.g., assay during purification of enzymes or for measurement of phenylalanine hydroxylase activity in the liver of hyperphenylalaninemics.The method is based on the phenylalanine-dependent change in absorbance of the tetrahydropteridine cofactor as it is oxidized to the dihydro form. The reaction rate measured by this procedure is linear over a wide range of enzyme concentration. The K_m and V for both tetrahydropteridine and for phenylalanine were the same as the values determined by the old procedure. Measurement of the stoichiometry of the reaction showed that one dihydropteridine is formed per tyrosine formed, or per DPNH consumed. The rate of reaction was identical to that measured by a coupled assay using DPNH and purified dihydropteridine reductase.     

Thermostable Flavin Reductase That Couples with Dibenzothiophene Monooxygenase,from Thermophilic Bacillus sp. DSM411: Purification,Characterization, and Gene Cloning

Flavin reductase is essential for the oxygenases involved in microbial dibenzothiophene (DBT) desulfurization. An enzyme of the thermophilic strain, Bacillus sp. DSM411, was selected to couple with DBT monooxygenase (DszC) from Rhodococcus erythropolis D-1. The flavin reductase was purified to homogeneity from Bacillus sp. DSM411, and the native enzyme was a monomer of M_r 16 kDa. Although the best substrates were flavin mononucleotide and NADH, the enzyme also used other flavin compounds and acted slightly on nitroaromatic compounds and NADPH. The purified enzyme coupled with DszC and had a ferric reductase activity. Among the flavin reductases so far characterized, the present enzyme is the most thermophilic and thermostable. The gene coded for a protein of 155 amino acids with a calculated mass of 17,325 Da. The enzyme was overproduced in Escherichia coli, and the specific activity in the crude extracts was about 440-fold higher than that of the wild-type strain, Bacillus sp. DSM411.     

Antidiabetic activity of phenolic compounds from Pecan bark in streptozotocin-induced diabetic rats

The n-butanol fraction (BF) of bark of Pecan tree, Carya illinoinensis (Wangenh) K. Koch (Juglandaceae) afforded two new flavonol methyl ether: caryatin-3′ sulfate (6) and caryatin-3′ methyl ether-7-O-β-d-glucoside (7) while five known phenolics (1–5) were isolated from its ethyl acetate fraction (EAF). The structures of isolated compounds were established based on 1D and 2D NMR spectroscopy. The isolated compounds were investigated for their hypoglycaemic, antioxidant as well as the aldose reductase (AR) inhibitory effect in lenses of streptozocin diabetic rats. All the isolated compounds showed significant hypoglycaemic and antioxidant activities, except 5 and 6. A marked AR-inhibitory effect was identified for compounds 2, 3 and 7.     

Inhibition of dihydropteridine reductase by catecholamines and related compounds

Catecholamines and related compounds, such as dopamine, 5- or 6-hydroxydopamine, N-methyldopamine, tyramine, octopamine, norepinephrine and epinephrine, inhibit human liver dihydropteridine reductase (NADH:6,7-dihydropteridine oxidoreductase, EC 1.6.99.10) noncompetitively with Ki values ranging from 7.0 X 10(-6) - 1.9 X 10(-4)M (I50 values = 2.0 X 10(-5) - 2.0 X 10(-4)M). The tyrosine analogs alpha-methyltyrosine and 3-iodotyrosine are weak inhibitors of this enzyme (I50 greater than 10(-3)M). The inhibitory effect of catecholamines is slightly decreased by O-methylation of one hydroxyl group, but is essentially abolished by total methylation. The inhibitory strength of the catecholamines and related compounds tested against this enzyme can be arranged in the following order: dopamine, 6-hydroxydopamine, 5-hydroxydopamine, N-methyldopamine greater than tyramine, 3-O-methyldopamine, 4-O-methyldopamine much greater than epinephrine, 3-O-methylepinephrine, norepinephrine, octopamine less than tyrosine much less than alpha-methyltyrosine, 3-iodotyrosine much less than homoveratrylamine. These results suggest that dopamine, norepinephrine and epinephrine may serve as physiological regulators of mammalian dihydropteridine reductase.     

Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase

A barley (Hordeum vulgare L.) mutant, nar1a (formerly Az12), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from nar1a was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in nar1a differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nar1a were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. The nitrate reductase from nar1a exhibits greater NADPH than NADH activity and has apparent K_m values for nitrate and NADH that are approximately 10 times greater than those of the wild-type enzyme. The nar1a nitrate reductase has apparent K_m values of 170 micromolar for NADPH and 110 micromolar for NADH. NADPH, but not NADH, inhibited the enzyme at concentrations greater than 50 micromolar.     

The distribution of synaptosomal oxidative enzymes after exposure to deoxycholate

Abstract— The distributions of NADH₂ dehydrogenase, NADH, cytochrome c reductase and cytochrome oxidase have been determined utilizing synaptosomal isolation techniques. Deoxycholate was used to determine compartmentation and/or ‘latency’ of these activities. NADPH, dehydrogenase proved to be a soluble and mitochondrial enzyme and the activity of this enzyme was not appreciably changed by deoxycholate treatment. NADH_g cytochrome c reductase proved to be a mitochondrial enzyme with considerable activity in microsomal fractions. Deoxycholate treatment increased activity in the synaptosomal fraction 8.3-fold. A bimodal activation pattern was observed with synaptosomal and mitochondrial NADH, cyrochrome c reductase upon exposure to increasing concentrations of deoxycholate, with enhancement of activity at 0.25 % (w/v) and 0.50 % (w/v) deoxycholate. The enzyme was stable at concentrations of deoxycholate less than 0.25% (w/v) but was irreversibly inactivated at concentrations higher than 0.25% (w/v). The mechanism of this activation pattern appeared to be a combination of enzyme release and inactivation. Similar results were not observed in liver mitochondria. Cytochrome oxidase, a known mitochondrial marker, exhibited a 17-fold increase in synaptosomal activity with deoxycholate treatment. The synaptosomal cytochrome oxidase activity after deoxycholate treatment approached the activity in the free mitochondrial fraction. The percentage of mitochondrial protein in synaptosomal fractions was estimated to be about 30 per cent from a comparison of the respective total (deoxycholate-treated) activities. On the basis of these data we suggest that the synaptosomal fraction possesses a relatively sizable energy-producing potential which may be of significance in vivo.     

Purification and immunochemical characteristics of NADPH-cytochrome P-450 oxidoreductase from tobacco cultured cells

NADPH-cytochrome P-450 oxidoreductase (EC 1.6.2.4) was purified from the microsomal fraction of tobacco (Nicotiana tabacum) BY2 cells by chromatography on two anion-exchange columns and 2′,5′ ADP-Sepharose 4B column. The purified enzyme showed a single protein band with a molecular weight of 79 kDa on SDS-PAGE and exhibited a typical flavoprotein redox spectrum, indicating the presence of an equimolar quantity of FAD and FMN. This enzyme followed Michaelis-Menten Kinetics with K_m values of 24 μM for NADPH and 16 μM for cytochrome c. An in vitro reconstituted system of the purified reductase with a partially purified tobacco cytochrome P-450 preparation showed the cinnamic acid 4-hydroxylase activity at the rate of 14 pmol min ⁻¹nmol⁻¹ P-450 protein and with a purified rabbit P-450_2C14 catalyzed N-demethylation of aminopyrine at the rate of 6 pmol min⁻¹ lnmo⁻¹ P-450 protein. Polyclonal antibodies raised against the purified reductase reacted with tobacco reductase but not with yeast reductase on Western blot analysis. Anti-yeast reductase antibodies did not react with the tobacco reductase. This result indicate that the tobacco reductase was immunochemically different from the yeast reductase. The anti-tobacco reductase antibodies totally inhibited the tobacco reductase activity, but not the yeast reductase. Also, Western blot analyses using the anti-tobacco reductase antibodies revealed that leaves, roots and shoots of Nicotiana tabacum plants contained an equal amount of the reductase protein. From these results, it was suggested that there are different antibody binding sites, which certainly participate in enzyme activity, between tobacco and yeast reductase.     

Interrelationships among human aldo-keto reductase: immunochemical, kinetic and structural properties

We have propsed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α, β subunits, and aldehyde reductase II is a monomer of δ subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (α and β) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (α subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li₂SO₄(0.4 M) for the expression of maximum enzyme activity, and its K_m for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li₂SO₄ and its K_m for glucose is more than 200 mM. The β subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The β subunits hybridized with the α subunits of placenta aldehyde I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristics properties of placenta aldehyde reductase I.     

Inhibition of dihydropteridine reductase in rat striatal synaptosomes and from human liver by metabolites of biogenic amines

Catecholamines are potent noncompetitive inhibitors of dihydropteridine reductase in rat striatal synaptosomal preparations or purified from human liver. Their metabolites, except homovanillic acid, also inhibit the enzyme from both sources. The inhibitory potency of these compounds depends on the presence of the catechol or the 4-hydroxyphenyl structure, but may be modified by the 2-carbon side chain and its substituents. Indoleamines which have a hydroxylated aromatic nucleus (5-hydroxytryptamine and 5,6-dihydroxytryptamine) are equally inhibitory to the enzyme. These results suggest that biogenic amines themselves rather than their metabolites may serve as physiological inhibitors of dihydropteridine reductase in rat brain.     

Peptidomimetic 2-cyanopyrrolidines as potent selective cathepsin L inhibitors

Cathepsins have been found to have important physiological roles. The implication of cathepsin L in various types of cancers is well established. In a search for selective cathepsin L inhibitors as anticancer agents, a series of 2-cyanoprrolidine peptidomimetics, carrying a nitrile group as warhead, were designed. Two series of compounds, one with a benzyl moiety and a second with an isobutyl moiety at P₂ position of the enzyme were synthesized. The synthesized compounds were evaluated for inhibitory activity against human cathepsin L and cathepsin B. Although, none of the compounds showed promising inhibitory activity, (E)N-{(S)1-[(S)2-cyano-1-pyrrolidinecarbonyl]-3-methylbutyl}-2,3-diphenylacrylamide (24) with an isobutyl moiety at P₂ was found to show selectivity as a cathepsin L inhibitor (Ki 5.3 μM for cathepsin L and Ki > 100 μM for cathepsin B). This compound could act as a new lead for the further development of improved inhibitors within this inhibitor type.     

Synthesis of chiral pyrazolo[4,3-e][1,2,4]triazine sulfonamides with tyrosinase and urease inhibitory activity

A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine with chiral amino group has been synthesized and characterized. The compounds were tested for their tyrosinase and urease inhibitory activity. Evaluation of prepared derivatives demonstrated that compounds (8b) and (8j) are most potent mushroom tyrosinase inhibitors whereas all of the obtained compounds showed higher urease inhibitory activity than the standard thiourea. The compounds (8a), (8f) and (8i) exhibited excellent enzyme inhibitory activity with IC₅₀ 0.037, 0.044 and 0.042?μM, respectively, while IC₅₀ of thiourea is 20.9?μM.     

Inhibitors of Glutathione Reductase as Potential Antimalarial Drugs Kinetic Cooperativity and Effect of Dimethyl Sulphoxide on Inhibition Kinetics

Abstract

We have developed inhibitors of glutathione reductase that improve on the inhibition of literature lead compounds by up to three orders of magnitude. Thus, analogues of Safranine O and menadione were found to be strong, reversible inhibitors of yeast glutathione reductase. Safranine O exhibited partial, uncompetitive inhibition with K_i and α values of 0.5 mM and 0.15, respectively. Thionine O was a partial (hyperbolic) uncompetitive inhibitor with K_i and α values of 0.4μM and 0.15, respectively. LY83583 and 2-anilino-l,4-naphthoquinone also showed (hyperbolic) partial, uncompetitive inhibition with micromolar K_i values. For Nile Blue A a model for two-site binding with (parabolic) uncompetitive inhibition fitted the data with a K_i value of 11 μM and a kinetic cooperativity between the sites of 0.12, increased to 0.46 by pre-incubation of the enzyme and Nile Blue A in the presence of glutathione disulphide. Analysis of the effects of preincubation on the kinetics and cooperativity indicated the possibility of a slow conformational change in the homodimeric enzyme, the first such indication of kinetic cooperativity in the native enzyme to our knowledge. Further evidence of conformational changes for this enzyme came from studies of the effects of dimethyl sulphoxide which indicated that this co-solvent, which at low concentrations has no apparent effect on initial velocities under normal assay conditions, induced a slow conformational change in the enzyme. Thionine O, Nile Blue A and LY83583 were redox-cycling substrates producing superoxide ion, detectable by means of cytochrome c reduction, but leading to no loss of glutathione reductase activity, under aerobic or anaerobic conditions. The water-soluble Safranine analogues Methylene Blue, Methylene Green, Nile Blue A and Thionine O (5 mg/kg i.p. x 5) were effective antimalarial agents in vivo against P. berghei, but their effect was small and a higher dose (50mg/kg i.p. x 1) was toxic in mice. Comparison was made with human glutathione reductase and its literature-reported interactions with several tricyclic inhibitors as studied by X-ray diffraction. It is possible that the conformational changes detected in the present study from alterations in detailed kinetic inhibition mechanisms may shed light on information transfer through the glutathione reductase molecule from the dimer interface ligand pocket to the active-site.     

Physarum polycephalum expresses a dihydropteridine reductase with selectivity for pterin substrates with a 6-(1', 2'-dihydroxypropyl) substitution

Physarum polycephalum is one of few non-animal organisms capable of synthesizing tetrahydrobiopterin from GTP. Here we demonstrate developmentally regulated expression of quinoid dihydropteridine reductase (EC 1.6.99.7), an enzyme required for recycling 6,7-[8H]-dihydrobiopterin. Physarum also expresses phenylalanine-4-hydroxylase activity, an enzyme that depends on dihydropteridine reductase. The 24.4 kDa Physarum dihydropteridine reductase shares 43% amino acid identity with the human protein. A number of residues important for function of the mammalian enzyme are also conserved in the Physarum sequence. In comparison to sheep liver dihydropteridine reductase, purified recombinant Physarum dihydropteridine reductase prefers pterin substrates with a 6-(1', 2'-dihydroxypropyl) group. Our results demonstrate that Physarum synthesizes, utilizes and metabolizes tetrahydrobiopterin in a way hitherto thought to be restricted to the animal kingdom.     

Catalytic properties of NADPH-specific dihydropteridine reductase from bovine liver

The catalytic properties of a new type of dihydropteridine reductase, NADPH-specific dihydropteridine reductase [EC 1.6.99.10], from bovine liver, were studied and compared with those of the previously characterized enzyme, NADH-specific dihydropteridine reductase [EC 1.6.99.7]. With quinonoid-dihydro-6-methylpterin, approximate Km values of NADPH-specific dihydropteridine reductase for NADPH and NADH were estimated to be 1.4 micron and 2,900 microns, respectively. The Vmax values were 1.34 mumol/min/mg with NADPH and 1.02 mumol/min/mg with NADPH. With NADPH, the Km values of the enzyme for the quinonoid-dihydro forms of 6-methylpterin and biopterin were 1.4 micron and 6.8 microns, respectively. The enzyme was inhibited by its reaction product, NADP+, in a competitive manner, and the inhibition constant was determined to be 3.2 microns. The enzyme was severely inhibited by L-thyroxine and by 2,6-dichlorophenolindophenol.