The presence of glycollate oxidase and dehydrogenase in Dunaliella primolecta

Homogenates of Dunaliella primolecta, D. salina and D. tertiolecta were assayed for glycollate oxidase and glycollate dehydrogenase. Both D. primolecta and D. salina but not D. tertiolecta showed substantial glycollate-dependent O₂-uptake which is characteristic of glycollate oxidase. L-Lactate was an alternative substrate and both glycollate- and L-lactate-dependent O₂ uptake were insensitive to 2 mM cyanide. Glycollate dehydrogenase, measured by following the glycollate-dependent reduction of 2,6-dichlorophenolindophenol under aerobic conditions, was present in D. primolecta, D. salina and D. tertiolecta. In the presence of glycollate and D-lactate, rates were additive so both glycollate and D-lactate dehydrogenases are present in the homogenates. Glycollate and D-lactate oxidation were both inhibited by 2 mM cyanide. Organelles released from phototrophically grown cells of D. primolecta were separated by isopycnic centrifugation on sucrose gradients. Glycollate oxidase was present in the peroxisome fraction at an equilibrium density of 1.25 g/cm³, while the major peak of glycollate dehydrogenase activity was in the mitochondrial fraction at an equilibirium density of 1.22 g/cm³.     

Hydrogen peroxide production and the release of carbon dioxide during glycollate oxidation in leaf peroxisomes

Summary The rate at which H₂O₂ becomes available during glycollate oxidation for further oxidation reactions, especially that of glyoxylate to formate and CO₂, in peroxisomes from spinach-beet (Beta vulgaris L., var. vulgaris) leaves has been determined by measuring O₂ uptake in the presence and absence of added catalase. The rates observed under air and pure O₂ were sufficient to account for the ¹⁴CO₂ released from [l-¹⁴C]glycollate under these conditions; the two reactions showed similar characteristics. In the course of the reaction, a fall in catalase activity was observed concomitant with an increase in ¹⁴CO₂ release. There is no evidence that catalase was disproportionately lost from the peroxisomes during isolation, and it is argued that the CO₂ release observed contributes to the photorespiratory CO₂ loss in intact leaves.Abbreviations DCPIP 2,6-dichlorophenolindophenol - FMN Flavin mononucleotide     

Glycollate metabolism of wheat and rice leaves during senescence and under the influence of growth regulators

The glycollate metabolism of wheat (Triticum vulgare Vill. cv. Sonalika) and rice (Oryza sativa L. ev. Jaya) leaves was studied during senescence by estimating the endogenous levels of glycollate and hydrogen peroxide (H₂O₂) and the activities of glycollate oxidase and catalase. In comparison with light incubation the incubation of excised leaves in the dark caused a decline in the glycollate content and in the activities of glycollate oxidase and catalase, and an increase in the H₂O₂ content, more marked in the leaves of rice than in the leaves of wheat. Glycollate oxidase activity gradually decreased with incubation time, and glycollate metabolism decreased during senescence. The glycollate oxidase in particular and glycollate metabolism of rice were more sensitive to incubation time than those of wheat. Kinetin increased the glycollate oxidase activity and glycollate metabolism during senescence, while ethrel (2-chloroethylpho-sphonic acid) and ABA (abscisic acid) reduced these activities in both plant species.     

Glyoxylate decarboxylation during glycollate oxidation by pea leaf extracts: significance of glyoxylate and extract concentrations

Hydrogen peroxide-dependent glyoxylate decarboxylation occurring during glycollate oxidation by pea leaf extracts (Pisum sativum L.) has been studied in relation to the effects of glyoxylate and extract concentration. With a saturating concentration of glycollate, decarboxylation was greatly stimulated by raising the glyoxylate concentration; at 30°C and with approx. 0.04 nkat of glycollate oxidase (as leaf extract) in the reaction mixture, CO₂ release in the presence of 5 mM glycollate and 5 mM glyoxylate was equal to about 45% of glycollate oxidation. However, CO₂ release at these substrate concentrations was not linearly proportional to the amount of extract supplied and was equal to a diminishing proportion of glycollate oxidation as the amount of extract was increased. This was shown to be due to the low affinity of catalase for H₂O₂, so that the endogenous catalase was able to destroy a larger proportion of the H₂O₂ generated at higher extract concentrations. It is argued that although at high glycoxylate concentrations (5–10 mM) in vitro, glyoxylate decarboxylation can be made to equal more than a third of the glycollate oxidised, less than 10% of the glyoxylate generated in vivo is likely to be decarboxylated in peroxisomes where high concentrations of glycollate oxidase and catalase are localised and where high concentrations of glyoxylate are unlikely to be maintained.Abbreviation PHMS pyrid-2-yl--hydroxymethane sulphonic acid     

Bleaching of Dichlorophenolindophenol by a Coupled Oxidation with Linoleate Catalyzed by Soybean Lipoxygenase

The coupled bleaching of 2,6-dichlorophenolindophenol by soybean lipoxygenase-1, was found to occur only under anaerobic conditions with a characteristic lag phase quite unlike the wellknown induction phase associated with lipoxygenase-catalyzed oxidation of linoleate hydroperoxide (LOOH)—free linolelic acid. The duration of this distinctive lag phase was very sensitive to lipoxygenase concentrations and equalled the length of time required for the primary enzyme activity to render the reaction solution virtually anaerobic. The onset of bleaching was marked by a gradual build-up of a ketodiene presumably derived from LOOH. Singlet O₂ and superoxide anion did not appear to be involved in the enzyme- catalyzed bleaching while the xanthine-xanthine oxidase system known to produce O₂^? was effective in bleaching DCPIP. It is proposed that the bleaching reaction was a result of ah oxidative and irreversible alteration of DCPIP involving a number of reactive oxidants known to be produced anaerobically upon incubation of LOOH and linoleic acid with native lipoxygenase.     

Oxygen consumption by Campylobacter sputorum subspecies Bubulus with formate as substrate

The kinetics of oxygen utilization by the microaerophile Campylobacter sputorum subspecies bubulus was studied. With formate as substrate two enzyme systems were found to be responsible for electron transfer between formate and oxygen. In the case of lactate oxidation one enzyme system could account for the activity measured. One of the formateoxidizing systems possessed a high affinity for oxygen [K _m(O₂)=approx. 4M O₂]. From inhibitor studies it was concluded that a respiratory chain was involved in its activity. Respiration by this system must be responsible for proton translocation and electron transport-linked phosphorylation at formate oxidation. The other enzyme system had an extremely low affinity for oxygen [K _m (O₂)=approx. 1 mM O₂]. It was tentatively identified as the H₂O₂-producing formate oxidase previously found in C. sputorum. The H₂O₂ production by this enzyme is implicated in an explanation of the microaerophilic nature of C. sputorum. Sensitivity of formate dehydrogenase to H₂O₂ was demonstrated. The influence of the formate concentration on aerobic formate oxidation was determined. The pH- and temperature dependencies of oxygen uptake with formate as substrate were examined at airsaturation and at a low dissolved oxygen tension.Abbreviations TL medium tryptose-lactate medium - TF medium tryptose-formate medium - HQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - SHAM salicylhydroxamic acid - DCPIP 2,6-dichlorophenolindophenol     

Metabolism and decarboxylation of glycollate and serine in leaf peroxisomes

The linked utilization of glycollate and L-serine has been studied in peroxisomal preparations from leaves of spinach beet (Beta vulgaris L.). The generation of glycine from glycollate was found to be balanced by the production of hydroxypyruvate from serine and similarly by 2-oxoglutarate when L-glutamate was substituted for L-serine. In the presence of L-malate and catalytic quantities of NAD⁺, about 40% of the hydroxypyruvate was converted further to glycerate, whereas with substrate quantities of NADH, this conversion was almost quantitative. CO₂ was released from the carboxyl groups of both glycollate and serine. Since the decarboxylation of both substrates was greatly in creased by the catalase inhibitor, 3-amino-1,2,4-triazole, and abolished by bovine liver catalase, it was attributed to the nonenzymic attack of H₂O₂, generated in glycollate oxidation, upon glyoxylate and hydroxypyruvate respectively. At 25–30° C, about 10% of the glyoxylate and hydroxypyruvate accumulated was decarboxylated, and the release of CO₂ from each keto-acid was related to the amounts present. It is suggested that hydroxypyruvate decarboxylation might contribute significantly to photorespiration and provide a metabolic route for the complete oxidation of glycollate, the magnitude of this contribution depending upon the concentrations of glyoxylate and hydroxypyruvate in the peroxisomes.     

Two sources of endogenous hydrogen peroxide in Escherichia coli

Mechanisms of hydrogen peroxide generation in Escherichia coli were investigated using a strain lacking scavenging enzymes. Surprisingly, the deletion of many abundant flavoenzymes that are known to autoxidize in vitro did not substantially lessen overall H₂O₂ formation. However, H₂O₂ production diminished by 25–30% when NadB turnover was eliminated. The flavin‐dependent desaturating dehydrogenase, NadB uses fumarate as an electron acceptor in anaerobic cells. Experiments showed that aerobic NadB turnover depends upon its oxidation by molecular oxygen, with H₂O₂ as a product. This reaction appears to be mechanistically adventitious. In contrast, most desaturating dehydrogenases are associated with the respiratory chain and deliver electrons to fumarate anaerobically or oxygen aerobically without the formation of toxic by‐products. Presumably, NadB can persist as an H₂O₂‐generating enzyme because its flux is limited. The anaerobic respiratory enzyme fumarate reductase uses a flavoprotein subunit that is homologous to NadB and accordingly forms substantial H₂O₂ upon aeration. This tendency is substantially suppressed by cytochrome oxidase. Thus cytochrome d oxidase, which is prevalent among anaerobes, may diminish intracellular H₂O₂ formation by the anaerobic respiratory chain, whenever these organisms encounter oxygen. These two examples reveal biochemical and physiological arrangements through which evolution has minimized the rate of intracellular oxidant formation.     

Hydrogen production by photosystem I of Scenedesmus: Effect of heat and salicylaldoxime on electron transport and photophosphorylation

Summary Photosynthesis, photoreduction, the p-benzoquinone Hill reaction, and glucose uptake by whole cells, as well as cyclic photophosphorylation (with PMS) by chloroplast particles were strongly inhibited by 10^-2 M salicylaldoxime or by heating whole cells for 1–2 min at 55°. In contrast, H₂ photoproduction by whole cells of mutant No. 11 and wild type Scenedesmus and PS I-mediated MR reduction by chloroplast particles were either stimulated or not significantly inhibited by these agents. H₂ production by mutant No. 8 was slightly depressed by salicylaldoxime. DCMU inhibited H₂ photoproduction with 10^-2 M salicylaldoxime approximately 20%, indicating some contribution of electrons by endogenous organic compounds to photosystem II between the O₂-evolving mechanism and the DCMU-sensitive site. We conclude that photohydrogen production by PS I of Scenedesmus does not require cyclic photophosphorylation but is due to non-cyclic electron flow from organic substrate(s) through PS I to hydrogenase where molecular H₂ is released.The following abbreviations were used CI-CCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP dichlorophenol-indophenol - MR methyl red - PMS phenazine methosulfate - PS photosystem This work was supported by contract AT-(40-1)-2687 from the U.S. Atomic Energy Commission to Professor H. Gaffron.     

The Effect of Various Alkaline Salts on the Glycolate Oxidase of Salicornia europaea and Pisum sativum in vitro

The response of glycolate oxidase from shoots of Salicornia europaea L. and from leaves of Pisum sativum L. to salt treatment during assay was studied by DCPIP reduction and O₂ uptake. In Pisum there was found up to five times more glycolate oxidase activity per gram fresh weight than in Salicornia. However, the calculation of the specific activity pointed out that this result was caused only by the high level of enzyme protein in Pisum, and that specific activity from both species was of equal size. By the DCPIP method it was shown that in test media containing up to 1.0 M NaCl or KCl glycolate oxidase of Salicornia was of equal size compared with the control (medium without additional salts). With 2.0 M NaCl or KCl the activity decreased by about 80 and 30% respectively. Glycolate oxidase of Pisum was somewhat more salt sensitive. 1.0 M NaCl or KCl reduced the activity by about 35%. In the presence of 2.0 M NaCl or KCl the enzyme activity from Pisum was inhibited to about 80 and 60% respectively. By substituting sulfates for chlorides the activity of glycolate oxidase from both Salicornia and Pisum was stimulated strongly. 1.5 M Na₂SO₄ and 0.5 M K₂SO₄ (both are saturated solutions) caused an increase of glycolate activity from Salicornia of about 225 and 185% respectively, and from Pisum of about 50 and 30% respectively. Studying the response of glycolate oxidase to salt treatment by O₂ uptake one must establish that with this method the degree of inhibition of enzyme activity at higher salt concentrations was always more severe than with dye reduction. Addition of 1.0 M NaCl or KCl to the assay medium caused an inhibition of glycolate oxidase activity from Salicornia of about 50% and from Pisum of about 60%. 2.0 M NaCl or KCl reduced the enzyme activity of both Salicornia and Pisum to nearly 10% of control activity. Furthermore, in contrast to DCPIP reduction no stimulating effect of sulfates on glycolate oxidase activity was detectable. Indeed, the inhibitory effect of sulfates was very slight. 1.0 M Na₂SO₄ caused a mean inhibition of glycolate oxidase activity of only 15% with both species, and in the presence of 1.5 M Na₂SO₄ 50% of control activity was measured. At maximal K₂SO₄ concentrations (0.5 M) glycolate oxidase from both Salicornia and Pisum was also unaffected. It is supposed that the described salt tolerance of glycolate oxidase in vitro, possibly is due to an adaptation of the enzyme to high salt levels within peroxisomes in vivo.     

化能自养硫氧化细菌Halothiobacillus sp.LS2介导的以乙炔为电子受体的硫氧化反应

【目的】探究化能自养硫氧化细菌Halothiobacillus sp. LS2介导的以乙炔为电子受体的厌氧硫氧化反应。【方法】稀释涂布法测定细胞生长情况,离子色谱仪测试硫氧化动力学中SO_4~(2–)和S_2O_3~(2–)以及基于相对荧光定量法的基因表达分析。【结果】尽管菌株LS2在以氧气为电子受体时的最大反应速率V_(max)更高,但在厌氧条件下且以乙炔为电子受体时,菌株LS2的生长量是氧气为电子受体时的2倍,且硫氧化酶基因soxB的表达量显著高于氧气作为电子受体时。【结论】菌株LS2不仅可以以乙炔为电子受体完成厌氧硫氧化反应,且这一代谢过程的产能效率较有氧硫氧化过程更高。本研究首次发现了微生物介导的以乙炔为电子受体的厌氧硫氧化反应,对丰富硫的生物地球化学循环理论有积极意义。     

Unique antioxidant effects of herbal leaf tea and stem tea from Moringa oleifera L. especially on superoxide anion radical generation systems

ABSTRACT

This study aimed to investigate the unique antioxidative effects of Japanese moringa products, herbal leaf tea and stem tea, using established free radical assays, focusing on superoxide anion (O₂^?) radical generation systems. Hot-water extracts from moringa teas resulted in different but lower scavenging activities than Trolox in four synthetic free radical models. Interestingly, these extracts further showed higher O₂^? radical scavenging effects than Trolox in the phenazine methosulfate-NADH-nitroblue tetrazolium and xanthine oxidase assay systems. Incubating human neutrophils in the presence of these tea extracts rather than Trolox effectively suppressed cellular O₂^? radical generation. Among the eight known phenolic constituents of moringa leaves, caffeic acid and chlorogenic acid may be responsible for the O₂^–specific radical scavenging capacity stronger than that of Trolox. These results suggest that moringa herbal teas are a good source of natural antioxidants for preventing O₂^? radical-mediated disorders.

Abbreviations: O₂^?: superoxide anion; ROS: reactive oxygen species; H₂O₂: hydrogen peroxide; XOD: xanthine oxidase; DPPH: 1,1-diphenyl-2-picrylhydrazyl; ABTS⁺: 2,2′-azinobis(2-ethylbenzothiazoline-6-sulfonic acid) cation; CPZ⁺: chlorpromazine cation; PMS: phenazine methosulfate; NBT: nitroblue tetrazolium; PMA: phorbol 12-myristate 13-acetate     

Fluoride Inhibition of Respiration and Fermentation in Cultured Cells of Acer pseudoplatanus

Low concentrations of sodium fluoride severely inhibit anaerobic CO₂ evolution in Acer pseadoplatanus L. cells but have relatively little effect on aerobic respiration. The insensitivity of respiratory O₂ uptake to fluoride is due in part to the fact that fluoride reduces the intracellular pyruvate concentration to only a relatively minor extent under aerobic conditions, although it prevents the several fold increase in endogenous private which is normally brought about by anoxia. The respiratory insensitivity is also ascribable to the existence of a respiratory component which is unaffected by the decrease in endogenous private resulting from fluoride treatment. The extremely severe respiratory inhibition brought about by fluoride plus dinitrophenol is not appreciably relieved by exogenous private, indicating that this inhibition is the result of interference with the aerobic oxidation process per se and is not solely a consequence of glycolytic inhibition.     

Glycollate oxidation by thylakoids of the cyanobacteria Anabaena cylindrica,Nostoc muscorum and Chlorogloea fritschii

The intracellular distribution of glycollate dehydrogenase EC 1.2.1.17 has been investigated in extracts of the cyanobacteria (blue-green algae) Anabaena cylindrica, Nostoc muscorum and Chlorogloea fritschii. Most of the enzyme activity was associated with a chlorophyll-containing cell-free pellet, which also exhibited Photosystem I and II activities. Sucrose density gradient centrifugation of this washed pellet resulted in the formation of a green band within which maximal chlorophyll concentration and enzymic glycollate oxidation coincided. Antiserum raised to this fraction obtained from A. cylindrica inhibited glycollate dehydrogenase and Photosystem II activity. The data indicate that most of the cyanobacterial glycollate dehydrogenase is associated with the thylakoids and thus provide evidence for the dual role of these membranes in photosynthetic and respiratory processes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - DPC diphenylcarbazide     

Oxygen activation and defence against oxygen toxicity in a psychrophilic bacteroidaceae

When suddenly exposed to air the growth of the obligate anaerobic bacterium of the bacteroidaceae type, strain B6, continues for a few hours before coming to a complete stop. When air is shut off soon after growth has ceased, the organism is able to reestablish anaerobic conditions due to an ability to reduce O₂, and resumes normal growth after another few hours. The O₂ reducing ability of the organism is due to the presence in the cells of a particlebound NADH oxidase, a soluble NADPH oxidase and a soluble pyruvate oxidase. The two pyridine nucleotide oxidase reduce O₂ to H₂O₂, the pyruvate oxidase reduces O₂ to H₂O. Catalase and peroxidase were not detected in anaerobically grown cells. Kinetic studies with cell-free extracts showed that the pyruvate oxidase had a considerably greater affinity (smaller K _m) for O₂ and capacity (higher V _max) for O₂ reduction than the two other oxidases. It is postulated that the pyruvate oxidase acts as a scavenger for O₂, leading to the non-toxic reduction product H₂O, and thus functions as a defense mechanism against oxygen toxicity when the organism is exposed to aerobic condition.Abbreviations PY peptone-yeast extract - PYG PY-glucose - PN pyridine nucleotide - PNH reduced PN - CCCP carbonylcyanide m-chlorophenylhydrazone - DNP 2.4-dinitrophenol     

Anaerobic Elemental Sulfur Reduction by Fungus Fusarium oxysporum

Reduction of inorganic sulfur compounds by the fungus Fusarium oxysporum was examined. When transferred from a normoxic to an anoxic environment, F. oxysporum reduced elemental sulfur to hydrogen sulfide (H₂S). This reaction accompanied fungal growth and oxidation of the carbon source (ethanol) to acetate. Over 2-fold more of H₂S than of acetate was produced, which is the theoretical correlation for the oxidation of ethanol to acetate. NADH-dependent sulfur reductase (SR) activity was detected in cell-free extracts of the H₂S-producing fungus, and was found to be up-regulated under the anaerobic conditions. On the other hands both O₂ consumption by the cells and cytochrome c oxidase activity by the crude mitochondrial fractions decreased. These results indicate that H₂S production involving SR was due to a novel dissimilation mechanism of F. oxysporum, and that the fungus adapts to anaerobic conditions by replacing the energy-producing mechanism of O₂ respiration with sulfur reduction.     

o-Diphenol: Oxygen oxidoreductase from leaves of sugar cane

A non-particulate o-diphenol: O₂ oxidoreductase (phenolase) has been isolated from leaves of sugar cane. Gel filtration produced two fractions MW 32000 and 130000. The preferred substrate was chlorogenic acid. Other o-diphenols (caffeic acid, catechol, pyrogallol, dihydroxyphenylalanine) all of which were slowly oxidized when tested alone, increased the rates of O₂ consumption obtained with catalytic amounts of chlorogenic acid. Both enzyme fractions were inhibited by thiols; thioglycollate, which acted in a non-competitive manner, was most effective.     

Anodic oxidation of coumarie and caffeic acids and their effects on nitrate uptake and nitrate reducta se inNicotia na tabacum cell suspension

Anodic oxidation of coumaric acid led to the inhibition of the process at the electrode due to a film which was formed after one-electron oxidation of the acid to phenoxy radical.By contrast, caffeic acid is oxidized in two steps-the phenoxy radical is formed in the first step, quinone in the second step. The inhibition of nitrate uptake by coumaric and caffeic acids is dependent on their concentration. 10^-4 M eaffeic acid totally inhibited nitrate uptake and the growth ofNicotiana tabacum cell suspension. 10^-6 M caffeic acid markedly inhibited nitrate uptake especially in the first three days after inoculation. 10^-6 M coumaric acid did not affect nitrate uptake and nitrate reductase activity, 10^-4 M coumaric acid inhibited nitrate uptake by day two after inoculation. Nitrate reductase synthesis correlated with the inhibition of nitrate uptake. Differential effects of coumaric and caffeic acids are explained on the basis of different products of their electrochemical oxidation.     

Inhibition of human catechol-O-methyltransferase (COMT)-mediated O-methylation of catechol estrogens by major polyphenolic components present in coffee

In the present study, we investigated the inhibitory effect of three catechol-containing coffee polyphenols, chlorogenic acid, caffeic acid and caffeic acid phenethyl ester (CAPE), on the O-methylation of 2- and 4-hydroxyestradiol (2-OH-E₂ and 4-OH-E₂, respectively) catalyzed by the cytosolic catechol-O-methyltransferase (COMT) isolated from human liver and placenta. When human liver COMT was used as the enzyme, chlorogenic acid and caffeic acid each inhibited the O-methylation of 2-OH-E₂ in a concentration-dependent manner, with IC₅₀ values of 1.3–1.4 and 6.3–12.5 μM, respectively, and they also inhibited the O-methylation of 4-OH-E₂, with IC₅₀ values of 0.7–0.8 and 1.3–3.1 μM, respectively. Similar inhibition pattern was seen with human placental COMT preparation. CAPE had a comparable effect as caffeic acid for inhibiting the O-methylation of 2-OH-E₂, but it exerted a weaker inhibition of the O-methylation of 4-OH-E₂. Enzyme kinetic analyses showed that chlorogenic acid and caffeic acid inhibited the human liver and placental COMT-mediated O-methylation of catechol estrogens with a mixed mechanism of inhibition (competitive plus noncompetitive). Computational molecular modeling analysis showed that chlorogenic acid and caffeic acid can bind to human soluble COMT at the active site in a similar manner as the catechol estrogen substrates. Moreover, the binding energy values of these two coffee polyphenols are lower than that of catechol estrogens, which means that coffee polyphenols have higher binding affinity for the enzyme than the natural substrates. This computational finding agreed perfectly with our biochemical data.     

Enzymes of glycollate formation and oxidation in two members of the rhodospirillaceae (Purple non-sulphur bacteria)

1. Phototrophic cultures of Rhodomicrobium vannielii do not excrete glycollate when gassed anaerobically with nitrogen plus carbon dioxide, although the addition of α-hydroxy-2-pyridine methanesulphonate (HPMS) results in the excretion of a trace amount of glycollate. The inclusion of low amounts of oxygen in this gas mixture results in marked glycollate excretion, higher rates occurring in the presence of HPMS.
2. Cell extracts of Rhodomicrobium vannielii, and also of Rhodospirillum rubrum, which excretes glycollate only under aerobic conditions in the light, catalyze the formation of glycollate from phosphoglycollate and also the oxidation of glycollate to glyoxylate.