Glutathione peroxidase in lens and a source of hydrogen peroxide in aqueous humour

1. Glutathione peroxidase has been demonstrated in cattle, rabbit and guineapig lenses. 2. The enzyme will oxidize GSH either with hydrogen peroxide added at the start of the reaction or with hydrogen peroxide generated enzymically with glucose oxidase. 3. No product other than GSSG was detected. 4. Oxidation of GSH can be coupled with oxidation of malate through the intermediate reaction of glutathione reductase and NADPH₂. 5. Traces of hydrogen peroxide are present in aqueous humour: it is formed when the ascorbic acid of aqueous humour is oxidized. 6. Hydrogen peroxide will diffuse into the explanted intact lens and oxidize the contained GSH. The addition of glucose to the medium together with hydrogen peroxide maintains the concentration of lens GSH. 7. Glutathione peroxidase in lens extracts will couple with the oxidation of ascorbic acid. 8. It is suggested that, as there is only weak catalase activity in lens, glutathione peroxidase may act as one link between the oxygen of the aqueous humour and NADPH₂.     

A monophenol oxidase activity in extracts of sorghum

A p-hydroxycinnamic acid oxidase activity was present in enzyme preparations from first internodes of Sorghum vulgare variety Wheatland milo when incubated in phosphate buffer at pH 7.5. This preparation had no classical polyphenolase activity but had both peroxidase and catalase activities. Since horseradish preparations catalyzed the same reaction, the oxidation probably is another example of a peroxidase-oxidase reaction. A second substrate was p-hydroxyphenylpyruvic acid. Ferulic acid was slightly active at low concentrations and inhibitory at higher ones. Diphenols such as caffeic and chlorogenic acids were inactive and inhibitory to p-hydroxycinnamic acid oxidation. A variety of monophenols such as tyrosine and cinnamic acid were inactive. An active substrate must have a free monophenolic group and para to this a C₃ side chain with a double bond and probably a free terminal acid group. A sulfhydryl reducing agent at the 5 millimolar level such as mercaptoethanol, reduced glutathione, or dithiothreitol was obligatory. Products were varied and were found in both the ethyl acetate-soluble and insoluble fractions after acidification of the incubation mixtures. With internode extracts, about 1 micromole of O₂ was consumed per micromole of p-hydroxycinnamic acid that disappeared in the presence of mercaptoethanol. Tetrahydrafolic acid plus mercaptoethanol were required for a second step oxidation or a parallel reaction; about 2 micromoles of O₂ were consumed per micromole of p-hydroxycinnamic acid that disappeared. Potassium cyanide, diethyldithiocarbamate, ascorbic acid, and ethylenediaminetetraacetate were inhibitory. A similar mercaptoethanol-dependent monophenol oxidase was present in preparations from green shoots that also contained a classical polyphenolase activity. The activity was present in both soluble and particulate (500 to 100,000 gravity) fractions of internodes. Preliminary studies were made of enzyme complexes in the particulate fractions capable of converting phenylalanine and tyrosine to the level of ferulic acid when the above p-hydroxycinnamic acid oxidase was blocked with ascorbic acid. The ratelimiting step was the hydroxylation of p-hydroxycinnamic acid.     

Effect of some reducing agents on water stress-induced oxidative and deteriorative processes ofVigna seedlings

Decreasing substrate osmotic potential produced in seedlings ofVigna catjang Endl. (cv. Pusa Barsati) proportional decrease in relative water content and leaf water potential, increase in respiration rate, proline content, H₂O₂ content, and the activities of indole acetic acid oxidase, ascorbic acid oxidase, peroxidase and glycolate oxidase but decrease in catalase activity and glycolate content. Pretreatment with reducing agents like L-cysteine or reduced glutathione (10?3 M) caused lower decrease in the relative water content, leaf water potential and glycolate content and reduced the rise of respiration rate, proline content and H₂O₂ content and also the activities of aforementioned oxidative enzymes, except catalase activity which was increased. Such treatments also maintained the chlorophyll and protein levels and decreased the tissue permeability. It was concluded that the treatment ofVigna seedlings with reducing agents reduced the deteriorative changes and oxidative processes which are characteristic of water stressed tissue.     

Changes in the Ascorbate System during Seed Development of Vicia faba L

Large changes occur in the ascorbate system during the development of Vicia faba seed and these appear closely related to what are generally considered to be the three stages of embryogenesis. During the first stage, characterized by embryonic cells with high mitotic activity, the ascorbic acid/dehydroascorbic acid ratio is about 7, whereas in the following stage, characterized by rapid cell elongation (stage 2), it is lower than 1. The different ascorbic/dehydroascorbic ratio may be correlated with the level of ascorbate free radical reductase activity, which is high in stage 1 and lower in stage 2. Ascorbate peroxidase activity is high and remains constant throughout stages 1 and 2, but it decreases when the water content of the seed begins to decline (stage 3). In the dry seed, the enzyme disappears together with ascorbic acid. Ascorbate peroxidase activity is observed to be 10 times higher than that of catalase, suggesting that ascorbate peroxidase, rather than catalase, is utilized in scavenging the H₂O₂ produced in the cell metabolism. There is no ascorbate oxidase in the seed of V. faba. V. faba seeds acquire the capability to synthesize ascorbic acid only after 30 days from anthesis, i.e. shortly before the onset of seed desiccation. This suggests that (a) the young seed is furnished with ascorbic acid by the parent plant throughout the period of intense growth, and (b) it is necessary for the seed to be endowed with the ascorbic acid biosynthetic system before entering the resting state so that the seed can promptly synthesize the ascorbic acid needed to reestablish metabolic activity when germination starts.     

24-Epibrassinolide alleviated zinc-induced oxidative stress in radish (Raphanus sativus L.) seedlings by enhancing antioxidative system

This article encompasses the results on the effects of 24-epibrassinolide (EBR) on the changes in reactive oxygen species (ROS) and activities of antioxidative enzymes in radish (Raphanus sativus L.) seedlings subjected to zinc (Zn) stress. Zn toxicity resulted in significant enhancement in the level of membrane lipid peroxidation, protein oxidation, contents of hydrogen peroxide (H₂O₂) and hydroxyl radical (^·OH), the production rate of superoxide radicals (O ₂ ^·? ) and the activities of lipoxygenase and NADPH oxidase in radish seedlings indicating the induction of oxidative stress. However, Zn-mediated enhancement in indices of oxidative stress was considerably decreased by EBR treatment. EBR application enhanced the activities of catalase, superoxide dismutase, guaiacol peroxidase, glutathione peroxidase, and peroxidase in radish seedlings under Zn stress. EBR treatment reduced the activity of ascorbic acid oxidase in Zn stressed seedlings. Further, EBR application also enhanced the free proline and phenol levels under Zn stress. From the results obtained in this study, it can be inferred that EBR application alleviated oxidative damage caused by over production of ROS through the up regulation of antioxidative capacity in Zn stressed radish seedlings.     

Regulation of glutamine synthetase by metal-catalyzed oxidative modification in the marine oxyphotobacterium Prochlorococcus

The inactivation of glutamine synthetase (GS; EC 6.3.1.2) by metal-catalyzed oxidation (MCO) systems was studied in several Prochlorococcus strains, including the axenic PCC 9511. GS was inactivated in the presence of various oxidative systems, either enzymatic (as NAD(P)H+NAD(P)H-oxidase+Fe³⁺+O₂) or non-enzymatic (as ascorbate+Fe³⁺+O₂). This process required the presence of oxygen and a metal cation, and is prevented under anaerobic conditions. Catalase and peroxidase, but not superoxide dismutase, effectively protected the enzyme against inactivation, suggesting that hydrogen peroxide mediates this mechanism, although it is not directly responsible for the reaction. Addition of azide (an inhibitor of both catalase and peroxidase) to the MCO systems enhanced the inactivation. Different thiols induced the inactivation of the enzyme, even in the absence of added metals. However, this inactivation could not be reverted by addition of strong oxidants, as hydrogen peroxide or oxidized glutathione. After studying the effect of addition of the physiological substrates and products of GS on the inactivation mechanism, we could detect a protective effect in the case of inorganic phosphate and glutamine. Immunochemical determinations showed that the concentration of GS protein significantly decreased by effect of the MCO systems, indicating that inactivation precedes the degradation of the enzyme.     

Evidence for H2O2 as the product of reduction of oxygen by alternative oxidase in mitochondria from potato tubers

Oxygen consumption by alternative oxidase (AOX), present in mitochondria of many angiosperms, is known to be cyanide-resistant in contrast to cytochrome oxidase. Its activity in potato tuber (Solanum tuberosum L.) was induced following chilling treatment at 4 °C. About half of the total O₂ consumption of succinate oxidation in such mitochondria was found to be sensitive to SHAM, a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive oxygen consumption by nearly half, and addition at the end of the reaction released nearly half of the consumed oxygen by AOX, both typical of catalase action on H₂O₂. These findings with catalase suggest that the product of reduction of AOX is H₂O₂ and not H₂O, as previously surmised. In potatoes subjected to chill stress (4 °C) for periods of 3, 5 and ?8 days the activity of AOX in mitochondria increased progressively with a corresponding increase in the AOX protein detected by immunoblot of the protein.     

Reaction Properties of the Ascorbic Acid Oxidase from Myrothecium verrucaria

Ascorbic acid oxidase activity in Myrothecium verrucaria extracts resulted in O(2) uptake exceeding 0.5 mole per mole of ascorbic acid and in CO(2) evolution. Measurement of oxidized ascorbic acid at completion of the reaction demonstrated that an average of 10% of the oxidized product disappeared. A comparison of the gas exchange data with the amount of ascorbic acid not accounted for indicated that the reaction could not be explained by independent oxidase and oxygenase systems. Chromatographic examination of the reaction mixtures identified l-threonic acid. Experiments with ascorbic acid-1-(14)C showed that C-1 was partially decarboxylated during the oxidation. Test of the fungal extracts for enzymes that might explain the deviation from expected stoichiometry showed that phenolase, glutathione reductase, cytochrome oxidase, peroxidase and oxalic decarboxylase were not involved. Addition of azide in concentrations sufficient to block catalase increased excess O(2) consumption about 65%. No enzymes were found that could directly attack oxidized ascorbic acid. H(2)O(2) accumulated during oxidation in azide-blocked systems.The O(2) excess could be explained by assuming the enzyme had peroxidative capacity on a reductant other than ascorbic acid. An intermediate of ascorbic acid oxidation appeared to function as the substrate yielding CO(2) and l-threonic acid on degradation. The increase in excess O(2) utilized in azide-blocked systems and the H(2)O(2) accumulation also were explained by the proposed scheme.Another interpretation would involve production of free radicals during ascorbic acid oxidation. Evidence for this was the ability of extracts to oxidize DPNH in the presence of ascorbic acid. Oxygen radicals formed in such reactions were considered possible agents of degradation of ascorbic acid.     

The role of superoxide and singlet oxygen in lipid peroxidation promoted by xanthine oxidase

The peroxidative oxidation of extracted rat liver microsomal lipid, assayed as malondialdehyde production, can be promoted by milk xanthine oxidase in the presence of 0.2 mM FeCl₃ and 0.1 mM EDTA. The reaction is inhibited by the superoxide dismutase activity of erythrocuprein. The reaction is also inhibited by 1,3-diphenylisobenzofuran, which reacts with singlet oxygen to yield dibenzoylbenzene. During inhibition of the lipid peroxidation reaction by 1,3-diphenylisobenzofuran, o-dibenzoylbenzene was produced. The rate of superoxide production by xanthine oxidase was not affected by 1,3-diphenylisobenzofuran. Lipid peroxidation promoted by ascorbic acid is not inhibited by either erythrocuprein or 1,3-diphenylisobenzofuran. Therefore it is suggested that the peroxidative oxidation of unsaturated lipid promoted by xanthine oxidase involves the formation of singlet oxygen from superoxide, and the singlet oxygen reacts with the lipid to form fatty acid hydroperoxides.     

Tea catechol oxidase: Isolation,purification and kinetic characterization

Cathechol oxidase extracted from tea leaves was purified over 200-fold, using isoelectric focusing. The purified catechol oxidase was free of peroxidase and flavanol gallate esterase activities. Further, this enzyme was shown to have optimum activity near pH 5·7 and a K_m of 2·3 × 10^?3 M (at 25°) for (?)-epigallocatechin gallate. The purified enzyme was found to be capable of epimerizing tea flavanols at their C-2 position whether oxidation of the flavanol occurs (aerobic conditions) or not (anaerobic conditions). When oxygen is present, gallic acid is formed as a result of oxidation of either (?)-epigallocatechin gallate or (?)-epicatechin gallate. Formation of gallic acid is a side reaction of the oxidation of the flavanol gallates and is named oxidative degallation; no esterase per se is involved in this reaction.     

Oxidation of methyl α-d-galactopyranoside by galactose oxidase: products formed and optimization of reaction conditions for production of aldehyde

The reaction conditions of galactose oxidase-catalyzed, targeted C-6 oxidation of galactose derivatives were optimized for aldehyde production and to minimize the formation of secondary products. Galactose oxidase, produced in transgenic Pichia pastoris carrying the galactose oxidase gene from Fusarium spp., was used as catalyst, methyl α-d-galactopyranoside as substrate, and reaction medium, temperature, concentration, and combinations of galactose oxidase, catalase, and horseradish peroxidase were used as variables. The reactions were followed by ¹H NMR spectroscopy and the main products isolated, characterized, and identified. An optimal combination of all the three enzymes gave aldehyde (methyl α-d-galacto-hexodialdo-1,5-pyranoside) in approximately 90% yield with a substrate concentration of 70 mM in water at 4 °C using air as oxygen source. Oxygen flushing of the reaction mixture was not necessary. The aldehyde existed as a hydrate in water. The main secondary products, a uronic acid (methyl α-d-galactopyranosiduronic acid) and an α,β-unsaturated aldehyde (methyl 4-deoxy-α-d-threo-hex-4-enodialdo-1,5-pyranoside), were observed for the first time to form in parallel. Formation of uronic acid seemed to be the result of impurities in the galactose oxidase preparation. ¹H and ¹³C NMR data of the products are reported for the α,β-unsaturated aldehyde for the first time, and chemical shifts in DMSO-d₆ for all the products for the first time. Oxidation of d-raffinose (α-d-galactopyranosyl-(1-6)-α-d-glucopyranosyl-(1-2)-β-d-fructofuranoside) in the same optimum conditions also proceeded well, resulting in approximately 90% yield of the corresponding aldehyde.     

Colorimetric alcohol assays with alcohol oxidase

A method is described for manufacturing crude alcohol oxidase (EC 1.1.3.13) preparations which are suitable for application in colorimetric alcohol assays. The procedure involves a one-step removal of catalase activity from a partially purified preparation of alcohol oxidase from the yeast Hansenula polymorpha via dialysis against 3-amino-1,2,4-triazole and hydrogen peroxide. Thus, the irreversible inactivation of more than 90% of the catalase present was achieved, which is prerequisite for the use of alcohol oxidase preparations in colorimetric alcohol assays via peroxidase-mediated oxidation of dyes. This type of assay was shown to be rapid, accurate and sensitive. The influence of the relative concentrations of the various assay constituents such as alcohol oxidase, catalase and peroxidase is discussed. It is concluded that this colorimetric alcohol assay is particularly suitable for the determination of ethanol in fermentation broths, both in qualitative and in quantitative tests.     

The oxidation of lysine and oxalysine by Mytilus edulis: Identification of the products formed in the presence and the absence of catalase

1. O-(2-Aminoethyl)serine (oxalysine) was shown to be a substrate of the l-amino acid oxidase of the digestive gland of the common mussel, Mytilus edulis. 2. Three atoms of oxygen were consumed per mole of oxalysine oxidized in the presence of catalase; l-lysine under the same conditions consumed only one atom. 3. The products of oxidation of oxalysine in the presence and the absence of catalase were: ethanolamine, N-oxalylethanolamine and 3-morpholone (the oxygen analogue of 2-piperidone). After acid hydrolysis 70% of the oxalysine oxidized was recovered as ethanolamine. 4. In the absence of catalase 2-aminoethoxyacetic acid was also detected. 5. The products identified account quantitatively for the oxalysine oxidized and for the oxygen uptake. 6. N-Oxalylethanolamine and 2-aminoethoxyacetic acid have been synthesized. 7. Treatment of extracts of the digestive gland at pH3·0 completely inactivated the catalase, leaving the l-amino acid oxidase unaffected. 8. The major product of the oxidation of lysine in the absence of catalase was 2-piperidone.     

An enzymatic component of the insect blood-brain barrier: Implications of DAB (3,3′-diaminobenzidine) oxidation

The oxidation of DAB (3,3′-diaminobenzidine) was examined in intact brains of the European corn borer, Ostrinia nubilalis. The deposition of oxidized DAB was examined by electron microscopy and found to be localized in glial cells. A semi-quantitative comparison of this reaction was made in vitro between horseradish peroxides, catalase, and insect brain. Inhibition characteristics indicate that the brain reaction is not the result of an endogenous peroxidase or catalase. The use of specific inhibitors (i.e. sesamex, tranylcypromine) indicate that this reaction could be attributed to an amine oxidase (monoamine: O₂ oxido-reductase (deaminating), E.C. 1.4.3.4) of microsomal origin.     

Anaerobic stopped-flow studies of indole-3-acetic acid oxidation by dioxygen catalysed by horseradish C and anionic tobacco peroxidase at neutral pH: catalase effect

The effect of order of reagent mixing in the absence and in the presence of catalase on the transient kinetics of indole-3-acetic acid (IAA) oxidation by dioxygen catalysed by horseradish peroxidase C and anionic tobacco peroxidase at neutral pH has been studied. The data suggest that haem-containing plant peroxidases are able to catalyse the reaction in the absence of exogenous hydroperoxide. The initiation proceeds via the formation of the ternary complex enzyme-->IAA-->oxygen responsible for IAA primary radical generation. The horseradish peroxidase-catalysed reaction is independent of catalase indicating a significant contribution of free radical processes into the overall mechanism. This is in contrast to the tobacco peroxidase-catalysed reaction where the peroxidase cycle plays an important role. The transient kinetics of IAA oxidation catalysed by tobacco peroxidase exhibits a biphasic character with the first phase affected by catalase. The first phase is therefore associated with the common peroxidase cycle while the second is ascribed to native enzyme interaction with skatole peroxy radicals yielding directly Compound II.     

The manganese toxicity of cotton

Cotton plants (Gossypium hirsutum. Linn. var. Sankar 4) were grown at normal and toxic levels of substrate manganese, and the altered metabolism of manganese toxic plants was studied. The tissues of plants exposed to toxic levels of manganese had higher activities of peroxidase and polyphenol oxidase, and the activities of catalase, ascorbic acid oxidase, glutathione oxidase and cytochrome c oxidase were lowered. In addition, the high manganese tissue had lower contents of ATP and glutathione but higher amounts of ascorbic acid. The respiration of the partially expanded leaves and the growing tips of toxic plants were depressed when compared to that of the normal tissues. The metabolic changes of manganese toxicity of cotton are placed in the following order: accumulation of manganese in the leaf tissue; a rise in respiration; stimulation of polyphenol oxidase; the appearance of initial toxicity symptoms; the evolution of ethylene and stimulation of peroxidase; the presence of severe toxicity symptoms; the depression of terminal oxidases and respiration; abscission of the growing tip and proliferation of the stem tissue. The early stimulation of polyphenol oxidase may be used to detect potential manganese toxicity.     

Microbial Consortium-Induced Changes in Oxidative Stress Markers in Pea Plants Challenged with Sclerotinia sclerotiorum

The ability for rhizobacteria and fungus to act as bioprotectants via induced systemic resistance has been demonstrated, and considerable progress has been made in elucidating the mechanisms of plant–biocontrol agent–pathogen interactions. Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27, and Bacillus subtilis BHHU100 from rhizospheric soils were used singly and in consortium and assessed on the basis of their ability to provide disease protection by relating changes in ascorbic acid and hydrogen peroxide (H₂O₂) production, lipid peroxidation, and antioxidant enzymes in pea under the challenge of Sclerotinia sclerotiorum. Increased production of H₂O₂ 24 h after pathogen challenge was observed and was 254.4 and 231.7–287.7 % higher in the triple consortium and singly treated plants, respectively, when compared to untreated challenged control plants. A similar increase in ascorbic acid content and ascorbate peroxidase activity was observed 24 and 48 h after pathogen challenge, respectively, whereas increased activities of catalase, guaiacol peroxidase, and glutathione peroxidase were observed 72 h after pathogen challenge. Similarly, lipid peroxidation reached a maximum at 72 h of pathogen challenge and was 61.2 and 11.2–32.1 % less in the triple consortium and singly treated plants, respectively, when compared to untreated challenged control plants. These findings suggest that the interaction of microorganisms in the rhizosphere enhanced protection from oxidative stress generated by pathogen attack through induction of antioxidant enzymes and improved reactive oxygen species management.     

Analysis of the peroxidatic mode of action of catalase

Catalase is an enzyme which can function either in the catabolism of hydrogen peroxide or in the peroxidatic oxidation of small substrates such as ethanol, methanol, or elemental mercury (Hg0). It has been reported that native catalase can peroxidatically oxidize larger organic molecules (e.g. L-dopa) and that catalase maintained at alkaline pH for various lengths of time demonstrates an increase in peroxidase activity using guaiacol as substrate. We have shown, by using two distinct methods of H2O2 introduction for measuring peroxidase activity, that native catalase shows no peroxidatic activity toward these larger organic molecules. We have also shown, through the use of these peroxidase assays and by enzyme absorption spectra, that the peroxidase activity attributed to catalase maintained at alkaline pH is a catalytic but not enzymatic activity associated with a hematin group attached to a denatured catalase monomer. Possible mechanisms for the catalytic and peroxidatic modes of action of catalase involving hydride-ion transfer are discussed.     

THE KINETICS OF THE DECOMPOSITION OF PEROXIDE BY CATALASE

It has been shown that the experimental results obtained by Morgulis in a study of the decomposition of hydrogen peroxide by liver catalase at 20°C. and in the presence of an excess of a relatively high concentration of peroxide are quantitatively accounted for by the following mechanisms. 1. The rate of formation of oxygen is independent of the peroxide concentration provided this is greater than about 0.10 M. 2. The rate of decomposition of the peroxide is proportional at any time to the concentration of catalase present. 3. The catalase undergoes spontaneous monomolecular decomposition during the reaction. This inactivation is independent of the concentration of catalase and inversely proportional to the original concentration of peroxide up to 0.4 M. In very high concentrations of peroxide the inactivation rate increases. 4. The following equation can be derived from the above assumptions and has been found to fit the experiments accurately. See PDF for Equation in which x is the amount of oxygen liberated at the time t, A is the total amount of oxygen liberated (not the total amount available), and K is the inactivation constant of the enzyme.     

THE DECOMPOSITION OF HYDROGEN PEROXIDE BY LIVER CATALASE

1. The velocity of decomposition of hydrogen peroxide by catalase as a function of (a) concentration of catalase, (b) concentration of hydrogen peroxide, (c) hydrogen ion concentration, (d) temperature has been studied in an attempt to correlate these variables as far as possible. It is concluded that the reaction involves primarily adsorption of hydrogen peroxide at the catalase surface. 2. The decomposition of hydrogen peroxide by catalase is regarded as involving two reactions, namely, the catalytic decomposition of hydrogen peroxide, which is a maximum at the optimum pH 6.8 to 7.0, and the "induced inactivation" of catalase by the "nascent" oxygen produced by the hydrogen peroxide and still adhering to the catalase surface. This differs from the more generally accepted view, namely that the induced inactivation is due to the H₂O₂ itself. On the basis of the above view, a new interpretation is given to the equation of Yamasaki and the connection between the equations of Yamasaki and of Northrop is pointed out. It is shown that the velocity of induced inactivation is a minimum at the pH which is optimal for the decomposition of hydrogen peroxide. 3. The critical increment of the catalytic decomposition of hydrogen peroxide by catalase is of the order 3000 calories. The critical increment of induced inactivation is low in dilute hydrogen peroxide solutions but increases to a value of 30,000 calories in concentrated solutions of peroxide.