Assay of UDP, GDP, CDP, and ADP reductase activities by column chromatography on polyethyleneimine cellulose

Deoxyribonucleosides were separated from ribonucleosides by chromatography on polyethyleneimine cellulose columns (Pasteur pipettes. The deoxyribonucleosides were quantitatively eluted with 25 mM boric acid in less than 10 ml while the ribonucleosides were retained. The ribonucleosides were eluted with 1 M NaCl. This method was utilized to assay for GDP, UDP, ADP, and CDP reductase activities after hydrolysis of the substrate and product nucleotides to the corresponding nucleosides. All four reductase activities were assayed using identical conditions of column size, eluting solution (25 mM boric acid), and elution volume. The use of polyethyleneimine cellulose columns with boric acid can be adapted to other enzyme assays such as purine nucleoside phosphorylase and for the isolation of deoxyribonucleotides from cellular extracts.     

Interaction of sulfate assimilation with nitrate assimilation as a function of nutrient status and enzymatic co-regulation inbrassica juncea roots

The interaction of sulfate assimilation with nitrate assimilation inBrassica juncea roots was analyzed by monitoring the regulation of ATP sulfurylase (AS), adenosine-5’-phosphosulfate reductase (AR), sulfite reductase (SiR), and nitrite reductase (NiR). Depending on the status of sulfur and nitrogen nutrition, AS and AR activities and mRNA levels were increased by sulfate starvation but decreased by nitrate starvation. The activation of AS and AR by sulfate starvation was inhibited by sulfate/nitrate starvation. However, the rise in steady-state mRNA levels for AS and AR by sulfate starvation was not affected by sulfate/nitrate starvation. SiR gene expression was slightly activated by both sulfate starvation and sulfate/nitrate starvation, but was decreased by nitrate starvation. Although NiR gene expression was little affected by sulfate starvation, it was diminished significantly by either nitrate or nitrate/sulfate starvation. Cysteine (Cys) also decreased AS and AR activities and mRNA levels even when plants were simultaneously starved for sulfate; in contrast, both SiR and NiR gene expressions were only slightly, if at all, affected under the same conditions. This supports our conclusion that Cys, the end-product of sulfate assimilation, is the key regulatory signal. Moreover, SiR and NiR apparently are not the linking step in the co-regulation of sulfate and nitrate assimilation in plants.     

Structural and functional analysis of denitrification genes in Pseudomonas stutzeri A1501

Denitrification is the process by which nitrates are converted to nitrogen gas under the action of microor-ganism, and in a bioenergetics viewpoint, a kind of respiration of bacteria in anoxia condition. In such a process, nitrogen in oxidation state replaces oxygen as the terminal electron acceptor in cell membrane, gen-erates potential gradient with the action of a series of oxidoreductase, and finally converts nitrate into nitro-gen[1]. Denitrification is widely present in nature, and resea…     

Chlorella vulgaris Aldehyde Reductase Is Capable of Functioning as Ferric Reductase and of Driving the Fenton Reaction in the Presence of Free Flavin

The free flavin-dependent Fenton reaction was detected in cell-free extracts of Chlorella. The corresponding enzyme was purified to homogeneity, and its N-terminal sequence was highly homologous to those of aldo-keto reductase family enzymes. The purified enzyme displayed aldehyde reductase activity in the presence of NADPH. Additionally, it showed ferric reductase activity and drove the Fenton reaction in the presence of free FAD and NADH.     

Microbial transformation of elements: the case of arsenic and selenium

Microbial activity is responsible for the transformation of at least one third of the elements in the periodic table. These transformations are the result of assimilatory, dissimilatory, or detoxification processes and form the cornerstones of many biogeochemical cycles. Arsenic and selenium are two elements whose roles in microbial ecology have only recently been recognized. Known as "essential toxins", they are required in trace amounts for growth and metabolism but are toxic at elevated concentrations. Arsenic is used as an osmolite in some marine organisms while selenium is required as selenocysteine (i.e. the twenty-first amino acid) or as a ligand to metal in some enzymes (e.g. FeNiSe hydrogenase). Arsenic resistance involves a small-molecular-weight arsenate reductase (ArsC). The use of arsenic and selenium oxyanions for energy is widespread in prokaryotes with representative organisms from the Crenarchaeota, thermophilic bacteria, low and high G+C gram-positive bacteria, and Proteobacteria. Recent studies have shown that both elements are actively cycled and play a significant role in carbon mineralization in certain environments. The occurrence of multiple mechanisms involving different enzymes for arsenic and selenium transformation indicates several different evolutionary pathways (e.g. convergence and lateral gene transfer) and underscores the environmental significance and selective impact in microbial evolution of these two elements. Electronic Publication     

Nitrate reduction in the wildtype and a nitrate reductase deficient mutant of Arabidopsis thaliana

A chlorate-resistant mutant B25 of Arabidopsis thaliana (L.) Heinh. was isolated, which has very little or no in vitro nitrate reductase activity and grows poorly on a substrate with nitrate as the sole nitrogen source. The mutation of B25 ( rgn ) is monogenic and recessive, tightly linked to the marker gene an on chromosome 1. Nitrate induces cytochrome- c reductase activity in the mutant but to a lower level than in the wildtype. After sucrose gradient centrifugation the greatest part of the cytochrome- c reductase from induced wildtype is found as 8s type whereas cytochrome- c reductase from B25 under the same conditions is found as 4s type. Nitrate reductase is found at the 8s position. It is suggested that B25 has lost the ability to assemble two 4s subunits showing cytochrome- c reductase activity and a Mo-bearing co-factor into the functional nitrate reductase. Nitrate rather than nitrite is the inducing agent for nitrite reductase, since in B25 nitrite reductase is even more rapidly induced than in the wildtype after addition of nitrate. Both the wildtype and B25 contain a nitrate reductase inhibiting factor when grown on ammonium. This inhibiting factor is a small protein, possibly similar to the nitrate reductase inactivating enzyme reported for other plants.     

Effects of artificially enhanced levels of ascorbate and glutathione on the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase in spinach (Spinacia oleracea)

Effect of high intracellular concentrations of the antioxidants ascorbate and glutathione on the extractable activity of the reducting enzymes dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase were investigated with spinach cells ( Spinacia oleracea ). An elevated ascorbate concentration was obtained by treatment with the ascorbate biosynthesis precursor L-galactono-1,4-lactone (GAL). To increase the intracellular level of glutathione, cells were treated with the 5-oxo-L-proline analog L-2-oxothiazolidin-4-carboxylate (OTC), or with the peroxidative herbicide acifluorfen (sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid). Extractable monodehydroascorbate reductase activity increased in the presence of a high level of ascorbate or glutathione, and enzyme activity was at maximum when cells were treated with acifluorfen + OTC, or acifluorfen + GAL. Extractable dehydroascorbate reductase activity decreased when the intracellular concentration of glutathione was high and non-enzymatic reduction of dehydroascorbate by glutathione was the dominant reaction. Maximal decrease of enzyme activity was found in cells treated with acifluorfen + OTC. Extractable activity of glutathione reductase (GR) increased after treatment of cells with acifluorfen alone, or acifluorfen + OTC, but enzyme activity was unaffected by a high intracellular concentration of glutathione obtained by treatment of cells with OTC alone, or by treatment with acifluorfen + GAL. The degree of GR activation seemed to be controlled by several factors including inhibition by a high concentration of glutathione and possibly oxidative damage to the enzyme. Overall, the enzymes tested in this study, which provide the reduced forms of ascorbate and glutathione, were differently affected by high antioxidant levels.     

与氮转化有关的土壤酶活性对抑制剂施用的响应

利用室内模拟培养试验,研究好气条件下施用尿素后土壤脲酶、脲酸还原酶、亚硝酸还原酶和羟胺还原酶活性对脲酶抑制剂氢醌（HQ）与硝化抑制剂包被碳化钙（ECC）和双氰胺（DCD）组合（HQ ECC、HQ DCD）的响应、结果表明,HQ DCD组合与其它抑制剂处理相比能更有效地降低土壤脲酶活性,增加硝酸还原酶、亚硝酸还原酶、羟胺还原酶活性,不同处理土壤脲酶、亚硝酸还原酶和羟胺还原酶活性与土壤NH4^ 、NO3^-、NH3挥发和N2O排放速率间存在不同形式的显著相关关系：土壤脲酶、亚硝酸还原酶和羟胺还原酶活性之间存在不同形式的显著正相关关系。     

The use of dimethylsulfoxide as a solvent in enzyme inhibition studies: the case of aldose reductase

Aldose reductase (AR) is an enzyme devoted to cell detoxification and at the same time is strongly involved in the aetiology of secondary diabetic complications and the amplification of inflammatory phenomena. AR is subjected to intense inhibition studies and dimethyl sulfoxide (DMSO) is often present in the assay mixture to keep the inhibitors in solution. DMSO was revealed to act as a weak but well detectable AR differential inhibitor, acting as a competitive inhibitor of the L-idose reduction, as a mixed type of non-competitive inhibitor of HNE reduction and being inactive towards 3-glutathionyl-4-hydroxynonanal transformation. A kinetic model of DMSO action with respect to differently acting inhibitors was analysed. Three AR inhibitors, namely the flavonoids neohesperidin dihydrochalcone, rutin and phloretin, were used to evaluate the effects of DMSO on the inhibition studies on the reduction of L-idose and HNE.     

Tetrahydrobiopterin is synthesized from 6-pyruvoyl-tetrahydropterin by the human aldo-keto reductase AKR1 family members

Tetrahydrobiopterin (BH(4)) is a cofactor for aromatic amino acid hydroxylases and nitric oxide synthase. The biosynthesis includes two reduction steps catalyzed by sepiapterin reductase. An intermediate, 6-pyruvoyltetrahydropterin (PPH(4)) is reduced to 1(')-oxo-2(')-hydroxypropyl-tetrahydropterin (1(')-OXPH(4)) or 1(')-hydroxy-2(')-oxopropyl-tetrahydropterin (2(')-OXPH(4)), which is further converted to BH(4). However, patients with sepiapterin reductase deficiency show normal urinary excretion of pterins without hyperphenylalaninemia, suggesting that other enzymes catalyze the two reduction steps. In this study, the reductase activities for the tetrahydropterin intermediates were examined using several human recombinant enzymes belonging to the aldo-keto reductase (AKR) family and short-chain dehydrogenase/reductase (SDR) family. In the reduction of PPH(4) by AKR family enzymes, 2(')-OXPH(4) was formed by 3 alpha-hydroxysteroid dehydrogenase type 2, whereas 1(')-OXPH(4) was produced by aldose reductase, aldehyde reductase, and 20 alpha-hydroxysteroid dehydrogenase, and both 1(')-OXPH(4) and 2(')-OXPH(4) were detected as the major and minor products by 3 alpha-hydroxysteroid dehydrogenases (types 1 and 3). The activities of aldose reductase and 3 alpha-hydroxysteroid dehydrogenase type 2 (106 and 35 nmol/mg/min, respectively) were higher than those of the other enzymes (0.2-4.0 nmol/mg/min). Among the SDR family enzymes, monomeric carbonyl reductase exhibited low 1(')-OXPH(4)-forming activity of 5.0 nmol/mg/min, but L-xylulose reductase and peroxisomal tetrameric carbonyl reductase did not form any reduced product from PPH(4). Aldose reductase reduced 2(')-OXPH(4) to BH(4), but the other enzymes were inactive towards both 2(')-OXPH(4) and 1(')-OXPH(4). These results indicate that the tetrahydropterin intermediates are natural substrates of the human AKR family enzymes and suggest a novel alternative pathway from PPH(4) to BH(4), in which 3 alpha-hydroxysteroid dehydrogenase type 2 and aldose reductase work in concert.     

Identification of new potent inhibitor of aldose reductase from Ocimum basilicum

Recent efforts to develop cure for chronic diabetic complications have led to the discovery of potent inhibitors against aldose reductase (AKR1B1, EC 1.1.1.21) whose role in diabetes is well-evident. In the present work, two new natural products were isolated from the ariel part of Ocimum basilicum; 7-(3-hydroxypropyl)-3-methyl-8-β-O-d-glucoside-2H-chromen-2-one (1) and E-4-(6′-hydroxyhex-3′-en-1-yl)phenyl propionate (2) and confirmed their structures with different spectroscopic techniques including NMR spectroscopy etc. The isolated compounds (1, 2) were evaluated for in vitro inhibitory activity against aldose reductase (AKR1B1) and aldehyde reductase (AKR1A1). The natural product (1) showed better inhibitory activity for AKR1B1 with IC₅₀ value of 2.095 ± 0.77 µM compare to standard sorbinil (IC₅₀ = 3.14 ± 0.02 µM). Moreover, the compound (1) also showed multifolds higher activity (IC₅₀ = 0.783 ± 0.07 µM) against AKR1A1 as compared to standard valproic acid (IC₅₀ = 57.4 ± 0.89 µM). However, the natural product (2) showed slightly lower activity for AKR1B1 (IC₅₀ = 4.324 ± 1.25 µM). Moreover, the molecular docking studies of the potent inhibitors were also performed to identify the putative binding modes within the active site of aldose/aldehyde reductases.     

Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase

Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.     

Nitrate and nitrite reduction by alfalfa root nodules: Accumulation of nitrite in Rhizobium melioti bacteroids and senescence of nodules

Addition of NO⁻₃ rapidly induced senescence of root nodules in alfalfa ( Medicago sativa L. cv. Aragon). Loss of nodule dry matter began at the lowest NO⁻₃ concentration (10 m M ) but degradation of bacteroid proteins was only detected when nodules were supplied with NO⁻₃ concentrations above 20 m M .
Bacteroids from Rhizobium meliloti contained high specific activities of nitrate reductase (NR) and nitrite reductase (NiR). Both enzymes were presumably substrate-induced although substantial enzyme activities were present in the absence of NO⁻₃ Typical specific activities for soluble NR and NiR of bacteroids under NO⁻₃ free conditions were 1.2 and 1.4 μmol (mg protein)⁻¹h⁻¹, respectively. In the presence of NO⁻₃, the specific activity of NR was considerably greater than that of NiR, thus causing NO⁻₂ accumulation in bacteroids. Nitrite levels in the bacteroids were linearly correlated with specific activities of NR and NiR, indicating that NO⁻₂ is formed by bacteroid NR and that this NO⁻₂ in turn, induces bacteroid NiR. Accumulation of NO⁻₂ within bacteroids also indicates that NO⁻₂ inhibits nodule activity after feeding plants with NO⁻₃     

Effect of hydrostatic pressure on a mutant of Saccharomyces cerevisiae deleted in the trehalose-6-phosphate synthase gene

Mutants Saccharomyces cerevisiae deleted on the trehalose-6-phosphate synthase gene (tps1) and their parental wild-type cells were submitted to hydrostatic pressure in the range of 0–200 MPa. Experimental evidence showed that viability for both strains decreased with increasing pressure and that tps1 mutants, unable to accumulate trehalose, were more sensitive to hydrostatic pressure than the wild-type cells. Additionally, both tps1 and wild-type cells in the stationary phase, when there is an accumulation of endogenous trehalose, were more resistant to pressure than proliferating cells. Under these conditions, mutant cells were also more sensitive to pressure treatment than the wild type. The present work also showed that mild pressure pretreatment did not induce hydrostatic pressure resistance (barotolerance) in yeast cells.     

Isolation,sequence identification and tissue expression profile of two novel soybean (glycine max) genes-vestitone reductase and chalcone reductase

The complete mRNA sequences of two soybean (glycine max) genes-vestitone reductase and chalcone reductase, were amplified using the rapid amplification of cDNA ends methods. The sequence analysis of these two genes revealed that soybean vestitone reductase gene encodes a protein of 327 amino acids which has high homology with the vestitone reductase of Medicago sativa (77%). The soybean chalcone reductase gene encodes a protein of 314 amino acids that has high homology with the chalcone reductase of kudzu vine (88%) and medicago sativa (83%). The expression profiles of the soybean vestitone reductase and chalcone reductase genes were studied and the results indicated that these two soybean genes were differentially expressed in detected soybean tissues including leaves, stems, roots, inflorescences, embryos and endosperm. Our experiment established the foundation for further research on these two soybean genes.     

Effect of chilling on growth and nitrogen assimilation in Azolla caroliniana

Azolla caroliniana was exposed to 5 °C in darkness for 1, 2, 3, 5 or 7 d and then recovered for 7 d. Plants previously chilled for 2 or 3 d exhibited higher growth rates when transferred to normal temperature than either the control plants or those previously chilled for 5 or 7 d. Increased plant growth may be related to increased contents of chlorophyll, sucrose, and reducing sugars, due to increased photosynthetic capacity. In another experiment Azolla plants were chilled at 5 °C for 7 d and then transferred for 0, 4, 8, 12, or 16 d recovery to the N-free Hoagland solution or Hoagland solution containing 5 mM KNO₃. In previously chilled plants, the growth rate was decreased. In the medium supplemented with nitrogen, the growth rate was greater than in the N-free medium in both chilled and nonchilled plants. In chilled plants the decrease in growth rate may be related to the disturbance of Anabaena azollae cells where the protecting envelope of the heterocysts was deorganized. During the recovery the rate of N₂-fixation increased in both chilled and nonchilled plants up to 12 d after which both rates were similar. However, during the first 4 d the rate of the nonchilled plants was approximately 4-fold that of the previously chilled plants. Nitrate reductase and nitrite reductase activities in control plants were higher than in those previously chilled for 7 d. Both activities increased in nonchilled and previously chilled plants up to 12 d then decreased. The total protein content increased up to 12 d in chilled and nonchilled plants after which it decreased. Under all treatments, the values were higher in nonchilled plants than in those previously chilled ones and were also higher in presence of N than in its absence. Thus the presence of N-source in the medium counteracts the effect of chilling injury particularly during prolonged recovery.     

Aldose and aldehyde reductases in human tissues

Immunochemical characterizations of aldose reductase and aldehyde reductases I and II, partially purified by DEAE-cellulose (DE-52) column chromatography from human tissues, were carried out by immunotitration, using antisera raised against the homogenous preparations of human and bovine lens aldose reductase and human placenta aldehyde reductase I and aldehyde reductase II. Anti-aldose reductase antiserum cross-reacted with aldehyde reductase I, anti-aldehyde reductase I antiserum cross-reacted with aldose reductase and anti-aldehyde reductase II antiserum precipitated aldehyde reductase II, but did not cross-react with aldose reductase or aldehyde reductase I from all the tissues examined. DE-52 elution profiles, substrate specificity and immunochemical characterization indicate that aldose reductase is present in human aorta, brain, erythrocyte and muscle; aldehyde reductase I is present in human kidney, liver and placenta; and aldehyde reductase II is present in human brain, erythrocyte, kidney, liver, lung and placenta. Monospecific anti-α and anti-β antisera were purified from placenta anti-aldehyde reductase I antiserum, using immunoaffinity techniques. Anti-α antiserum precipitated both aldehyde reductase I and aldose reductase, whereas anti-β antibodies cross-reacted with only aldehyde reductase I. Based on these studies, a three gene loci model is proposed to explain the genetic interrelationships among these enzymes. Aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α and β subunits and aldehyde reductase II is a monomer of δ subunits.