Increase in production of ascorbate radical in tissues of rat treated with paraquat

The production of ascorbate radical (A^·-) was investigated in tissues of rats intoxicated with paraquat (PQ) to know the protective role of antioxidant ascorbate (AH^·-) in tissues. The electron spin resonance (ESR) method is applied to observe A^·-. To eliminate increased biosynthesis of ascorbic acid (AH₂) by PQ intoxication, ODS rats were chosen and fed with or without 250 ppm PQ in the diet. The radical A^·- was detected only in the lung and spleen homogenates of both intoxicated and control rats at the beginning of ESR measurement. The radical levels of intoxicated rat lung and spleen were increased rapidly to twice the initial level after 3 h and decreased to 0.2–0.6 times the initial level after 24 h, whereas those of control rats were increased slowly to 1.1 times the initial level after 4 h and decreased slowly to 0.7 times the initial level after 24 h at 4°C. In other organs such as liver, kidney, heart and testis, A^·- was not detected initially but detected afterwards. Higher A^·- level was observed in the intoxicated rat liver than the control but no appreciable differences of A^·- levels were observed between the intoxicated kidney, heart and testis and the respective controls. In the intoxicated rat lung the concentration of AH₂ is only half but that of A^·- is twice as high as that of the control. Larger amounts of A^·- produced in the intoxicated rats decayed more quickly than those in the control rats. The simple addition of PQ to the control organ enhanced neither A^·- production nor A^·- quenching. These facts suggest that the tissues damaged by PQ require larger amounts of AH^- to detoxicate harmful oxidants, resulting in concomitant production of A^·-.     

根系抗坏血酸在小麦幼苗铝耐性中的作用

以3个铝耐性不同的小麦品种为材料,研究了Al胁迫下小麦幼苗根系质外体和共质体抗坏血酸含量以及抗坏血酸氧化酶和过氧化物酶活性的变化。结果显示,Al耐性品种‘Atlas 66’质外体中抗坏血酸总含量随着处理 Al浓度的增加显著升高,而在Al敏感品种‘Scout 66’和‘扬麦9号’中显著降低。同时,‘Atlas 66’质外体中还原型抗坏血酸含量在高浓度Al处理下显著升高,2个敏感品种则在低浓度Al处理下还原型抗坏血酸含量略有升高。耐性品种‘Atlas 66’根系共质体还原型抗坏血酸和抗坏血酸总量在5～40μmol·L-1AlCl3处理下无显著变化,而在 2个敏感品种中则随处理Al浓度的增加显著下降。80μmol·L-1AlCl3处理下‘Atlas 66’根系质外体和共质体抗坏血酸氧化酶以及抗坏血酸过氧化物酶活性与对照相比均无显著变化,而在‘Scout 66’和‘扬麦9号’中则均显著降低。因此Al胁迫下‘Atlas 66’根系质外体抗坏血酸含量的升高和共质体抗坏血酸含量的维持以及Al毒害下抗坏血酸利用率较高可能是其Al耐性的一个重要机制。     

Vitamin C biosynthesis in prosimians: evidence for the anthropoid affinity of Tarsius

This report examines the taxonomic distribution of the in vitro biosynthesis of ascorbic acid in the Prosimii (Order: Primates). Liver and kidney samples of 15 prosimian taxa, including Tarsius bancanus, were quantitatively tested for the enzyme L-gulono-1,4-lactone oxidase. Liver samples from all taxa except Tarsius had substantial levels of the enzyme. Furthermore, unlike other eutherian mammals, kidney tissue from members of the family Lemuridae showed low but consistent levels of enzyme activity. The result for Tarsius, by fitting with the pattern exhibited by the monkeys, apes, and man, adds significant independent evidence for this animal's relatively close genetic relationship with the Anthropoidea.     

Biochemical characteristics of urban maple trees

The study, which covers the period between 2014 and 2018, was carried out in the city of Naberezhnye Chelny, Republic of Tatarstan, Russia. The aim of the study was to examine the biochemical response of maple trees growing in the anthropogenic environments. Leaf samples from 600 trees (Acer platanoides L. and Acer negundo L.) were collected at monthly intervals from June through August. Sampling was performed early in the morning (11 a.m.) in the middle of the month. The study offers statistical data on the tannin content, determined via permanganometry; the ascorbic acid concentration, found via titration with 2.6-dichlorophenolindophenol; the ascorbate oxidase activity determined by absorbance at 265 nm; and the polyphenol oxidase activity, found by the spectrophotometric method. Relatively higher ascorbate oxidase activity was detected in August among ash-leaved Acer platanoides L. and Acer negundo L. in areas with strong anthropogenic impact. Due to increased air pollution, maple trees were found to exhibit an increase of polyphenol oxidase activities. The condensed tannin content in Norway maple trees dropped over time: by 1.24 in July (avenue); by 0.94 (buffer area) and 0.76 (avenue) in August. The condensed tannin content in the ash-leaved maple trees also decreased: by 0.69 (buffer area) and 0.22 (avenue) in July; by 0.37 (buffer area) and 0.61(avenue) in August.     

二氧化硫污染对植物体酶活性的影响

通过用６种浓度的ＳＯ２气体对６种植物进行熏气处理,然后测定植物叶片内的过氧化物酶,抗坏血酸氧化酶及多酚氧化酶的活性,为环境监测提供理论依据。通过试验,发现ＳＯ２气体对植物叶内不同酶的活性的影响有别。６种植物受不同浓度的ＳＯ２污染,叶内的过氧化物酶活性随ＳＯ２浓度增加而增加,促进叶片的衰老;而对其他的２种酶,则６种植物表现不同     

Metabolic regulation of two types of phenol oxidase activity in ontogenesis of house fly

Changes of the tyrosinase activity in ontogenesis of the house fly Musca domestica were shown to be phase-specific and ontogenetic changes of tyrosinase and dihydroxyphenylalanine oxidase activities proved to be coordinated. Ascorbic acid stimulated some ontogenetic stages of the house fly and physiological indices, such as fertility, survival at different stages, and weight of puparia. Also, ascorbic acid modulated the tyrosinase activity.     

Kinetic Characterization of the Oxidation of Esculetin by Polyphenol Oxidase and Peroxidase

Esculetin has been described as an inhibitor of tyrosinase and polyphenol oxidase and, therefore, of melanogenesis. In this work, we demonstrate that esculetin is not an inhibitor but a substrate of mushroom polyphenol oxidase (PPO) and horseradish peroxidase (POD), enzymes which oxidize esculetin, generating its o-quinone. Since o-quinones are very unstable, the usual way of determining the enzymatic activity (slope of recordings) is difficult. For this reason, we developed a chronometric method to characterize the kinetics of this substrate, based on measurements of the lag period in the presence of micromolar concentrations of ascorbic acid. The catalytic constant determined was of the same order for both enzymes. However, polyphenol oxidase showed greater affinity (a lower Michaelis constant) than peroxidase for esculetin. The affinity of PPO and POD towards oxygen and hydrogen peroxide was very high, suggesting the possible catalysis of both enzymes in the presence of low physiological concentrations of these oxidizing substrates. Taking into consideration optimum pHs of 4.5 and 7 for POD and PPO respectively, and the acidic pHs of melanosomes, the studies were carried out at pH 4.5 and 7. The in vivo pH might be responsible for the stronger effect of these enzymes on L-tyrosine and L-3,4-dihydroxyphenylanaline (L-DOPA) (towards melanogenesis) and on cumarins such as esculetin towards an alternative oxidative pathway.     

Gulonolactone oxidase activity-dependent intravesicular glutathione oxidation in rat liver microsomes

The orientation of gulonolactone oxidase activity was investigated in rat liver microsomes. Ascorbate formation upon gulonolactone addition resulted in higher intravesicular than extravesicular ascorbate concentrations in native microsomal vesicles. The intraluminal ascorbate accumulation could be prevented or the accumulated ascorbate could be released by permeabilising the vesicles with the pore-forming alamethicin. The formation of the other product of the enzyme, hydrogen peroxide caused the preferential oxidation of intraluminal glutathione in glutathione-loaded microsomes. In conclusion, these results suggest that the orientation of the active site of gulonolactone oxidase is intraluminal and/or the enzyme releases its products towards the lumen of the endoplasmic reticulum.     

Net biosynthesis of antithrombin III by the isolated rat liver perfused for 12–24 hours compared with rat fibrinogen and α-2 (acute-phase) globulin,antithrombin III is not an acute phase protein

Antithrombin III-heparin cofactor has been isolated from normal rat plasma, purified to homogeneity on acrylamide gel electrophoresis and used to prepare a monospecific antiserum in rabbits. Measurements of rat antithrombin III were made by a single radial immunodiffusion assay.Net synthesis of antithrombin III was investigated during 12- or 24-h perfusions of the isolated rat liver. In perfusions performed under basal conditions cumulative synthesis of antithrombin-III was observed to occur at a rate sufficient to replace the total circulating plasma antithrombin III in about 6 h. In perfusions performed under full supplementation conditions which greatly enhanced synthesis of fibrinogen and α-2 (acute-phase) globulin (known acute-phase reactant proteins) net synthesis of antithrombin III was not significantly greater than that observed in control perfusions. Although these prolonged perfusion studies conclusively demonstrate net synthesis of antithrombin III by the isolated rat liver, they afford no evidence that this protein is an acute-phase reactant.     

柠檬酸和抗坏血酸对蝴蝶兰叶外植体褐变发生的影响

目的:探究柠檬酸和抗坏血酸对蝴蝶兰叶片外植体褐变发生的影响以及对PPO活性变化影响的作用机理.方法:以褐变率和褐变指数为参考数据,分析柠檬酸和抗坏血酸对外植体PPO活性和PPO反应产物积累的影响以及与外植体褐变发生的关系.结果:分别用100mg/L柠檬酸共培养和50mg/L抗坏血酸浸泡处理叶片外植体,经离体培养3d,褐变率分别比对照降低94.9%和54.9%,离体培养6d,褐变指数低于对照的0.53,分别为0.46和0.36,同时PPO活性降低.结论:推测柠檬酸抑制褐变的原因是直接与酶蛋白作用,抗坏血酸则与新生醌类物质结合.     

Effect of ethrel treatment on the postharvest changes in the turnover of ascorbic acid and reducing sugar inAchras sapota fruits

There was a definite relationship between growth and ascorbic acid content inAchras sapota. Increase in ethrel concentration from 250 ppm to 500 ppm hastened early ripening and increased the amount of reducing sugars but depleted the ascorbic acid content. Other aspects of ascorbic acid turnover viz. ascorbigen, bound form of ascorbic acid, ascorbic acid utilization, net ascorbic acid bound and ascorbic acid oxidase were also studied.     

Activities of Cu-containing proteins in Cu-depleted pea leaves

The effect of Cu deficiency on Cu-containing enzymes and on their activities was studied with two subsequent generations of Cu-deficient pea plants ( Pisum sativum L., cv. Progress) grown in low Cu²⁺ media. Cu deficiency caused growth inhibition and a decrease in photosynthesis as well as in the activities of 3 Cu-containing enzymes: diamine oxidase (EC 1.4.3.6), ascorbate oxidase (EC 1.10.3.3) and superoxide dismutase (EC 1.15.1.1). Determinations of photosynthetic electron-transport rates as well as the concentrations of several redox components showed that the target of Cu deprivation in the photosynthetic apparatus is the synthesis of Cu-containing plastocyanin which is positively correlated to the Cu content of the leaves. Inhibited formation of plastocyanin resulted in low activities of photosynthetic electron transport in photosystem I. Under Cu-deficient conditions, the activities of diamine oxidase and ascorbate oxidase were inhibited by about 50% in the first and 80% in the second generation of pea plants. Enzyme assays showed an inhibition of the activities of both the plastidic and cytoplasmic Cu/Zn-containing superoxide dismutases. An observed simultaneous increase of Mn-superoxide dismutase may be a compensation mechanism to partially maintain the total superoxide-dismutase activity under Cu-deficient conditions. This result indicates that the formation of superoxide-dismutase isoenzymes is interdependent and coordinated.     

Spectroscopic and binding studies of azide to type-2-copper-depleted ascorbate oxidase from zucchini

Summary Binding of azide to type-2-copper-depleted (T2D) zucchini ascorbate oxidase, containing reduced type-3 Cu centers, and met-T2D ascorbate oxidase, containing oxidized type-3 Cu centers, has been studied spectroscopically. In both cases titration with azide in 0.1 M phosphate pH 6.8 produces a broad near-ultraviolet band with maximum at 455 nm (e 2500 M^–1 cm^–1, with respect to the met-T2D enzyme) and shoulder at 390 nm (e 1700 M^–1 cm^–1), that are assigned to(azide)Cu(II) ligand-to-metal charge transfer (LMCT) transitions. This is accompanied by a reduction of absorbance at 330 nm in the met-T2D) enzyme adduct (e –1400 M^–1 cm^–1). A broad circular dichroic band of negative sign between 370–480 nm corresponds to the LMCT absorption band. Analysis of the titration data indicates that one azide ion binds independently to each of the binuclear T3 Cu couples with low affinity (K = 50 M^–1). The ESR signal of the T1 Cu observed in frozen solutions of the T2D enzyme is also perturbed by the addition of azide. The analogies in the azide-binding characteristics between ascorbate oxidase and laccase are discussed.     

Ascorbic Acid Oxidase: An Enzyme in Search of a Role

Ascorbic acid oxidase (AAO) has been fully characterized at molecular level, yet its functional role is unclear. The properties of the enzyme and the main hypotheses on its function are discussed. Recent data and reappraisal of previous observations suggest that AAO could be part of a dynamic mechanism operating whenever plant cells have to control oxygen availability. This revised version was published online in July 2006 with corrections to the Cover Date.     

Suppression of Powdery Mildew on Flax by Foliar Application of Essential Oils

Six essential oils were evaluated as to their efficiency in controlling powdery mildew (PM) of flax when they were applied as foliar sprays in an outdoor pot experiment. Onion, flax and fenugreek oils did not affect PM severity ratings – that is, they were ineffective in controlling the disease. On the other hand, black cumin, jojoba and coriander oil showed variable levels of efficiency in controlling the disease. Black cumin was moderately effective in controlling the disease because it reduced disease severity by 32.87%, while jojoba and coriander oils were highly effective as they reduced disease severity by 66.24 and 68.64%, respectively. Essential oils did not affect seed weight; however, coriander oil was a notable exception as it reduced seed weight by 55%. Straw weight was not affected by any oil. Foliar application of essential oils resulted in significant changes in the levels of protein, phenols, ascorbic acid and malondialdehyde and in activities of peroxidase and polyphenol oxidase. The lack of significant correlation between levels and activities of these biochemical components and PM severity demonstrate that these components are not involved in the suppression of PM by essential oils. Therefore, direct toxicity of essential oils to the causal pathogen Oidium lini is the most likely explanation for the disease suppression.     

Oxalic acid metabolism and calcium oxalate formation in Lemna minor L.

Abstract Axenic Lemna minor plants, which form numerous calcium oxalate crystals, were exposed to [¹⁴C]-glycolic acid, -glyoxylic acid, -oxalic acid and -ascorbic acid and prepared for microautoradiography by a technique that preserves only insoluble label to determine specifically the pathway leading to oxalic acid used for crystal formation. Label from glycolic, glyoxylic, and oxalic acids was incorporated into crystals. Label from oxalic acid was also found in starch when exposure to label was done in the light but not dark, while plastids specialized for lipid storage were heavily labelled under both conditions. Incorporation of label from glycolic and glyoxylic acids, but not oxalic acid, was inhibited in the presence of the glycolate oxidase inhibitors, αHPMS (2-pyridylhydroxy methanesulphonic acid) and mHBA (methyl 2-hydroxy-3-butynoic acid), and inhibition of labelling was not due to an effect on uptake. These studies show that the glycolate oxidase pathway to oxalic acid is operational in L. minor and that the product is available for crystal formation. Dark-grown plants form almost four times as many crystal cells (idioblasts) as do light-grown plants, indicating crystal formation is not in response to photorespiratory glycolate production. Label from [1-¹⁴C]ascorbic acid was also incorporated into crystals and labelling was inhibited by mHBA, indicating glycolic acid and/or glyoxylic acid are possible intermediates of ascorbic acid catabolism. The effect of nitrogen source on crystal formation was also investigated. Significantly more crystal idioblasts were formed, on a surface area basis, by plants grown on ammonium than by plants grown on nitrate nitrogen. When grown with mixed ammonium and nitrate, an intermediate number of crystal idioblasts were formed.     

Free radical mediated interaction of ascorbic acid and ascorbate/Cu(II) with viral and plasmid DNAs

Previous studies indicate that ascorbic acid, when combined with copper or iron cleaves several viral DNA. ln this study, we generated the ascorbate radical anion electrochemically in a simple chemical environment without the participation of a metal ion. This solution possesses viral DNA scission activity. Ohe absence of catalytic metal ions [Fe (III) and Cu(II)] in the incubation medium was evidenced by metal chelating agents such as desferrioxamine and EDTA. Ohe radical quenching at high EDTA concentration was attributed to ionic strength of EDTA rather than metal chelation. Ohe effects of antioxidants, radical scavangers, catalase, superoxide dismutase and some proteins on DNA cleavage have been tested. Cleavage may not arise directly from ascorbate free radical but the reaction of the radical form of ascorbate with oxygen may produce the actual reactive species. Aerobic oxidation of ascorbate itself strictly requires transition metal catalysts, however electrochemically produced ascorbyl radical avoided the kinetic barrier that prevented direct oxidation of ascorbic acid with oxygen and eliminated the need for the transition metal ion catalysts.     

The apoplastic antioxidant enzymatic system in the wood-forming tissues of trees

The complete apoplastic enzymatic antioxidant system, composed by class I ascorbate peroxidases (class I APXs), class III ascorbate peroxidases (class III APXs), ascorbate oxidases (AAOs), and other class III peroxidases (PRX), of wood-forming tissues has been studied in Populus alba, Citrus aurantium, and Eucalyptus camaldulensis. The aim was to ascertain whether these enzymatic systems may regulate directly (in the case of APXs), or indirectly (in the case of AAOs), apoplastic H₂O₂ levels in lignifying tissues, whose capacity to produce and to accumulate H₂O₂ is demonstrated here. Although class I APXs are particularly found in the apoplastic fraction of P. alba (poplar), and class III APXs are particularly found in the apoplastic fraction of C. aurantium (bitter orange tree), the results showed that the universal presence of AAO in the extracellular cell wall matrix of these woody species provokes the partial or total dysfunction of apoplastic class I and class III APXs, and of the whole plethora of non-enzymatic redox shuttles in which ascorbic acid (ASC) is involved, by the competitive and effective removal of ASC. In fact, the redox state (ASC/ASC+DHA) in intercellular wash fluids (IWFs) of these woody species was zero, and thus strongly shifted towards DHA (dehydroascorbate), the oxidized product of ASC. This imbalance of the apoplastic antioxidant enzymatic system apparently results in the accumulation of H₂O₂ in the apoplast of secondary wood-forming tissues, as can be experimentally observed. Furthermore, it is hypothesized that since AAO uses O₂ to remove ASC, it could regulate O₂ availability in the lignifying xylem and, thorough this mechanism, AAO could also control the activity of NADPH oxidase (the enzyme responsible for H₂O₂ production in lignifying tissues) at substrate level, by controlling the tension of O₂. That is, the presence of AAO in the extracellular cell wall matrix appears to be essential for finely tuning the oxidative performance of secondary wood-forming tissues.     

A frameshift mutation that elongates the penicillinase protein of Bacillus licheniformis

A penicillinase mutant penP102, isolated after ICR (acridine mustard) mutagenesis of Bacillus licheniformis strain 749/C, retains about 50% of the wild-type penicillinase specific activity. The penicillinase produced by this mutant differs from the wild-type protein in its sensitivity to pH and its electrophoretic behaviour. The penP102 mutation appears to have several other phenotypic effects, including an increase in the efficiency of release of the extracellular form of the enzyme.The penP102 penicillinase has been purified and its amino acid sequence compared to that of the wild-type enzyme. The mutation has resulted in the replacement of the last three amino acids of the wild-type enzyme and the addition of 17 residues at the carboxy-terminus. Comparison of the wild-type and mutant amino acid sequences shows that the mutational event is a single nucleotide deletion from the codon for asparagine²⁶⁵. Consideration of the possible nucleotide sequence for the region beyond the carboxy-terminus of the wild-type protein shows that there are no possible termination codons until four and six triplets beyond the codon for the carboxy-terminal lysine, indicating that the carboxy-terminus of the wild-type extracellular penicillinase is generated by proteolytic cleavage of a larger precursor protein.