Changes in spectral properties of ageing and senescing maize and sunflower leaves

Activity and biochemical characteristic of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase from pear ( Pyrus communis cv. Blanquilla) was determined. The enzyme showed a low K_m (57.5 μM) for ACC and was dependent on O₂ (K_m 0.44% in atmosphere). It had an absolute requirement for Fe²⁺, ascorbate and CO₂ and was inhibited by α-aminoisobutyric acid (AIB: K₁ 4.2 m M ) and cobalt. ACC oxidase has an optimum pH of 6.7 and temperature maxima at 28 and 38°C and it is concluded that the activity of ACC oxidase from pear resembles authentic in vivo activity.     

Peroxidase and polyphenol oxidase activities in Cyperus esculentus leaves following glyphosate applications

Following a 2-week treatment with glyphosate [N-(phosphonomethyl)glycine] changes in peroxidase (EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.14.18.1) activities of yellow nutsedge ( Cyperus esculentus L.) plants, were determined. Glyphosate caused significant increases of both activities. Isoelectric focusing gave 3 species (F₁, F₂ and F₃) of peroxidase activity, at pl 3.8, 4.4 and 4.8, and 4 species (F_a, F_b, F_c and F_d) of PPO activity at pl 7.0, 7.5, 7.8 and 9.5. The activity of the 4 active forms of PPO increased with increasing glyphosate dose up to 10⁻² M . The effect of the herbicide on the 3 fractions with peroxidase activity was to change their relative activities. Highest F₁ activity was found in control plants whereas the F₂ fraction was the predominant form in the plants treated with glyphosate at 10⁻² M and the highest F₃ activity occurred in plants treated with 5 × 10⁻³ M glyphosate. The increased PPO activity could produce phytotoxic o -quinones, and variations in peroxidase isoenzymes activity could enhance isoperoxidases with lignin biosynthetic activity.     

多酚氧化酶抑制剂对蝴蝶兰叶外植体褐变的影响

将多酚氧化酶(PPO)抑制剂添加到酶反应液中,抗坏血酸和半胱氨酸在0.5mmol/L就完全抑制蝴蝶兰PPO活性。300mg/L柠檬酸和100~200mg/L亚硫酸氢钠分别添加到培养基中,可使蝴蝶兰外植体褐变程度降低;采用抑制剂浸泡处理外植体,对外植体褐变抑制效果最好的为50mg/L抗坏血酸,外植体在褐变发生前PPO活性低于对照。     

Effect of dicarboxylic acids on the peroxidase-IAA oxidase isozymes of soybean callus

Multiple forms of peroxidase with indole-3-acetic acid (IAA) oxidase activity were detected in callus cultures from soybean seeds [ Glycinc max (L.) Merrill, cv. Acme] using ion-exchange chromatography and polyacrylamide gel electrophoresis. The properties of the IAA oxidase were studied with a partially purified fraction eluted from a DEAE cellulose column. At pH 5.7. p-coumaric acid and MnCl₂ were required as cofactors and H₂O₂ was not able to replace them, but H₂O₂ eliminated the usual lag period of the reaction. Activation effects obtained with some dicarboxylic acids acting only on IAA oxidase are shown. These effects were studied at different pH values and oxalic acid was found to be the most efficient activator, particularly at pH 4.6. Activation by oxalic acid occurred even in the absence of MnCl₂, but the presence of this salt produced a synergistic effect. IAA oxidase showed a sigmoidal kinetic behaviour at pH 5.7 changing to hyperbolic at pH 4.6     

Protective mechanisms of nitrogenase against oxygen excess and partially-reduced oxygen intermediates

Diazotrophic systems have developed a number of strategies to protect nitrogenase (N₂ase; EC 1.18.6.1) from O₂ excess and active-oxygen species (AOS). Protection against O₂ excess is given by biochemical modifications of N₂ase, increased rates of low-efficiency respiration, temporal segregation of N₂ fixation and photosynthesis, physical barriers to O₂ diffusion, and hemoglobins. On the other hand, AOS may originate from oxidation of N₂ase components, ferredoxins, flavodoxins and hemoglobins; interaction among the AOS themselves, or between H₂O₂ and hemoglobins; and during reactions catalyzed by hydrogenase (EC 1.18.99.1), xanthine oxidase (EC 1.1.3.22) and uricase (EC 1.7.3.3). Active-oxygen species are scavenged enzymatically [superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6). peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11)] or through non-enzymic reaction with low-molecular-weight compounds (ascorbate, α-tocopherol, glutathione).     

Prostaglandin H Synthetase-Mediated Metabolism of Dopamine: Implication for Parkinson's Disease

Abstract: Differences in prostaglandin H synthetase (PHS) activity in the substantia nigra of age- and post-mortem interval-matched parkinsonian, Alzheimer's, and normal control brain tissue were assessed. Prostaglandin E₂ (PGE₂, an index of PHS activity) was higher in substantia nigra of parkinsonian brain tissue than Alzheimer's or control tissue. Incubation of substantia nigra slices with arachidonic acid (AA) increased PGE₂ synthesis. Dopamine stimulated PHS synthesis of PGE₂. [³H]Dopamine was activated by PHS to electrophilic intermediate(s) that covalently bound to DNA, microtubulin protein, bovine serum albumin, and sulfhydryl reagents. When AA was replaced by hydrogen peroxide, PHS/H₂O₂-supported binding proceeded at rates similar to those observed with PHS/AA. Indomethacin and aspirin inhibited AA-mediated cooxidation of dopamine but not H₂O₂-mediated metabolism. PHS-mediated metabolism of dopamine was not affected by monoamine oxidase inhibitors. Substrate requirements and effects of specific inhibitors suggest cooxidation of dopamine is mediated by the hydroperoxidase activity of PHS. ³²P-postlabeling was used to detect dopamine-DNA adducts. PHS/AA activation of dopamine in the presence of DNA resulted in the formation of five dopamine-DNA adducts, i.e., 23, 43, 114, 70, and 270 amol/µg DNA. DNA adduct formation was PHS, AA, and dopamine dependent. PHS catalyzed cooxidation of dopamine in dopaminergic neuronal degeneration is discussed.     

EXTRACELLULAR PEROXIDASE-MEDIATED OXYGEN CONSUMPTION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

The unicellular green alga Chlamydomonas reinhardtii Dang. displays a high capacity for salicylhydroxamic acid (SHAM)—stimulated O₂ consumption, mediated by extracellular peroxidaie. Addition of exogenous NADH also resulted in stimulation of O₂ consumption. The SHAM-and NADH-stimulated peroxidase activity was partially sensitive to inhibition by exogenous superoxide dismutase, ascorbate, and gentisic acid. These compounds did not inhibit O₂ consumption in the absence of effectors. SHAM-and NADH-stimulated peroxidase activity also was sensitive to inhibition by cyanide, and cyanide titration curves indicated that O₂ consumption by peroxidase was more cyanide-sensitive than O₂ consumption by cytochrome oxidase. The differential sensitivity to cyanide was used to estimate partitioning of O₂ consumption between mitochondrial respiration and extracellular peroxidase. We suggest that, despite a large capacity for peroxidase-me-diated O₂ consumption, peroxidase did not consume O₂ at detectable rates in the absence of effectors. Therefore, in the absence of effectors, measured rates of O₂ consumption represented the rate of mitochondrial respiration .     

Characterization of extracellular oxygen consumption by the green alga Selenastrum minutum

Cells of the green alga Selenastrum minutum display a high capacity for extra-mitochondrial O₂ consumption in the presence of effectors such as salicylhydroxamic acid and/or NADH. We provide evidence that this O₂ consumption is mediated by extracellular peroxidase. Peroxidase capacity, measured as the potential for stimulation of O₂ consumption by a combination of salicylhydroxamic acid and NADH, changed over a 10-day time course. Maximal stimulation of O₂ consumption occurred at day three, at which point the capacity for peroxidase-mediated O₂ consumption was three-to four-fold higher than that of the control O₂ consumption rate. Peroxidase-mediated O₂ consumption was sensitive to inhibition by 50 m M ascorbate and by cyanide. Cyanide titration curves indicated that O₂ consumption by peroxidase was much more sensitive to inhibition by cyanide than was O₂ consumption by cytochrome oxidase (I₅₀ < 1.6 μ M and I₅₀= 18.3 μ M cyanide, respectively). By using evidence from a combination of cyanide titration curves and ascorbate inhibition, we concluded that despite a large capacity for peroxidase-mediated O₂ consumption, peroxidase did not measurably contribute to control rates of O₂ consumption. In the absence of effectors, O₂ consumption was mediated primarily by cytochrome oxidase.     

Monoamine Oxidase Activity of Mitochondria Prepared from Rat Liver and Rat Heart

Abstract: The effect of agents that change the respiratory state of the mitochondrion on tyramine oxidation was investigated. Neither uncoupler nor ADP and P_t in the presence of substrate produced any change in the rate of tyramine oxidation, as judged by direct measurement of tyramine oxidation or by H₂O₂ production. We conclude that previously reported depression of monoamine oxidase activity by stimulated respiration was due to oxygen depletion.     

Ascorbic Acid Inhibits 125I-SCH 23982 Binding but Increases the Affinity of Dopamine for D1 Dopamine Receptors

Abstract: Effects of ascorbic acid (AA) on ¹²⁵I-SCH 23982 binding to D₁ dopaminergic receptors in membrane preparations from rat striatum were investigated. AA in the range of 0.03 µ M –0.33 m M inhibited 75% of specific binding of ¹²⁵I-SCH 23982 in a dose-dependent manner. At higher concentrations, this inhibition of binding activity by AA was less potent, and 3.3 m M AA inhibited only 30% of specific binding. Reduced glutathione did not alter the inhibition of binding by 0.33 m M AA, but reduced the inhibition by 3.3 m M AA to 8% of specific binding. The loss of specific binding by AA was rescued by 1 m M EDTA, an inhibitor of lipid peroxidation. In the absence of AA, competition experiments with the agonist, dopamine, revealed the presence of high-affinity ( K _h = 224.9 ± 48.9 n M ) and low-affinity ( K _l = 21,100 ± 2,400 n M ) binding sites. Although the maximum binding of ¹²⁵I-SCH 23982 decreased to 40% without affecting the K _D value in the presence of 1.67 m M AA, the value of the high-affinity site for dopamine was increased ( K _h = 23.3 ± 9.4 n M ) and that of the low-affinity site was decreased ( K _l = 136,800 ± 40,900 n M ). These results suggest that AA may affect D₁ dopamine receptor function by lipid peroxidation, competition with dopamine for low-affinity sites, and reduced oxidation of dopamine.     

Catabolism of indole-3-acetic acid in citrus leaves: identification and characterization of indole-3-aldehyde oxidase

A new enzyme, named indole-3-aldehyde oxidase (IAldO), was identified in citrus ( Citrus sinensis L. Osbeck cv. Shamouti) leaves. The enzyme was partially purified by (NH₄)₂SO₄ fractionation. Sephadex G-200 gel filtration and DEAE-cellulose ion exchange chromatography. IAldO catalyzes the oxidation of indole-3-aldehyde (IAld) to indole-3-carboxylic acid (ICA) with the production of H₂O₂. The enzyme is highly specific for IAld. The apparent K_M of the enzyme for IAld is 19 μ M . The optimum oxidation of IAld occurs at pH 7. 5. The molecular mass of the enzyme, as determined by Sepharose-6B gel filtration, is about 200 kDa. Based on inhibitor studies, it is concluded that IAldO is not a flavin-linked oxidase and there is no requirement for free sulfhydryl groups or divalent cations for maximum activity. The enzyme is strongly inhibited by benzaldehyde. Ethylene pretreatment, wounding and aging of leaf tissues did not affect enzyme activity, suggesting that the enzyme is constitutive in citrus tissues.     

Azide-stimulated oxygen consumption by the green alga Selenastrum minutum

Dark O₂ consumption by the green alga Selenastrum minutum was sensitive to inhibition by the cytochrome pathway respiration inhibitor cyanide in the absence of an alternative oxidase inhibitor, consistent with previous work that suggested that this alga lacks alternative oxidase capacity. In contrast, addition of low concentrations of the cytochrome pathway inhibitor azide (50–750 μ M ) resulted in a stimulation of dark O₂ consumption, while higher concentrations of azide (1–2 m M ) partially inhibited O₂ consumption. Measurements of changes in cellular levels of pyruvate, malate and pyridine nucleotides upon cyanide addition were consistent with the absence of alternative oxidase capacity, and suggested that cyanide inhibition of O₂ consumption was not due to nonspecific effects of cyanide. Addition of salicylhydroxamic acid (SHAM) also resulted in an increase in the rate of O₂ consumption. Both azide- and SHAM-stimulated O₂ consumption were sensitive to inhibition by 50 m M ascorbate or by cyanide. However, the ubiquinone analogs chloroquine and quinacrine specifically inhibited azide-stimulated O₂ consumption, with only minor effects on SHAM-stimulated O₂ consumption. These results suggest that azide-stimulated O₂ consumption was not mediated by the previously characterized SHAM-stimulated oxidase, and are consistent with the possibility that azide-stimulated O₂ consumption is mediated by a plasma membrane redox system.     

Genetic manipulation of proline levels affects antioxidants in soybean subjected to simultaneous drought and heat stresses

It was assumed that the genetic manipulation of the proline (Pro) level would also affect the (homo)glutathione content as both compounds have a common precursor, glutamate. To test this hypothesis, the levels of Pro, reduced and oxidized (homo)glutathione [(h)GSH and (h)GSSG] and other antioxidants were compared under simultaneous drought and heat stress conditions and in a control treatment in a time course experiment on wild-type soybean ( Glycine max cv. Ibis) and on transgenic plants containing the cDNA coding for l -Δ¹-pyrroline-5-carboxylate reductase (EC 1.5.1.2), the last enzyme involved in Pro synthesis, in the sense and antisense directions. At the end of the recovery period, the highest H₂O₂ and lipid hydroperoxide concentrations were observed in the antisense transformants, which exhibited the greatest injury, while the lowest H₂O₂ content was detected in the sense transformants, which exhibited the lowest injury percentage. During stress treatment, the highest Pro and ascorbate (AA) levels were detected in the sense transformants, while the highest GSH and hGSH contents, AA/dehydroascorbate (DHA) and (h)GSH/(h)GSSG ratios and ascorbate peroxidase (APX) activity were found in the antisense transformants. The greatest APX (EC 1.11.1.11) activity was observed in the first part of the stress treatment in the antisense transformants, and the greatest glutathione reductase (EC 1.6.4.2) activity was observed in the second part of the treatment in the same genotype. The present experiments indicate that the manipulation of Pro synthesis affects not only the (h)GSH concentrations, but also the levels of other antioxidants.     

Characterization of polyphenol oxidase changes induced by desiccation of Ramonda serbica leaves

Resurrection plants are able to dehydrate/rehydrate rapidly without cell damage by a mechanism, the understanding of which may be of ecological importance in the adaptation of crop plants to dry conditions. The o -diphenol oxidase in Ramonda serbica Pan. & Petrov, a rare resurrection plant of the Balkan Peninsula, was characterized in respect to different isoforms, preferable substrates and specific inhibitors. Two anionic isoforms with pI 4.6 and 4.7 were separated from turgid leaves. Three additional anionic isoforms (pI 5.1, 5.3 and 5.6) and three neutral isoforms (pI from 6.8 to 7.4) were induced in desiccated leaves. Based on apparent K_m values, the affinity for reducing substrates decreased as follows: methyl catechol > chlorogenic acid > 3,4-dihydroxyphenylalanine > caffeic acid > pyrogallol. Polyphenol oxidase (PPO) activity was specifically sensitive to diethyldithiocarbamate and also inhibited by KCN, DTT and salicylic hydroxamic acid but with no inhibitory effect of Na₃N. Plants were subjected to drought-to-near complete water loss (approximately 2% relative water content, RWC) and several fold higher PPO activity was detected in desiccated leaves. Ramonda leaves contain high levels of phenolics, which decreased during drought. Rehydration of dry leaves from 2% RWC to 95% RWC led to transient inhibition of PPO in the first few hours. Within a day, the levels completely recovered to those determined in desiccated leaves. The finding of desiccation-induced high activity of PPO and new isoforms, which were also present in rehydrated turgid leaves, indicates a substantial role for PPO in the adaptation mechanism of resurrection plants to desiccation and also to the oxidative stress during rehydration.     

Arachidonic Acid Turnover and Phospholipase A2 Activity in Neuronal Growth Cones

Abstract: We analyzed de novo synthesis and local turnover of phospholipids in the growing neuron and the isolated nerve growth cone. The metabolism of phosphatidylinositol (PI) was studied with regard to the incorporation of saturated and unsaturated fatty acids and inositol. A comparison of de novo phospholipid synthesis in the intact neuron (whole brain, cell cultures) versus local turnover in isolated growth cone particles (GCPs) from fetal rat brain revealed different incorporation patterns and, in particular, high arachidonic acid (AA) turnover in PI of GCPs. These observations, together with elevated levels of free AA (2.5% of total AA content) in GCPs, demonstrate the predominance of acylation/deacylation in the sn -2 position of PI. GCP phospholipase A₂ (PLA₂) activity was demonstrated using [^3H]-or [¹⁴C]AA-phosphatidylcholine (PC) or -PI as the substrate in vitro and GCPs or a cytosolic GCP extract as the source of enzyme. In contrast to PC, which is hydrolyzed very slowly, PI is a very good GCP PLA₂ substrate. PLA₂ activity is much higher in GCPs than that of phospholipase C, as demonstrated by the comparison of AA and inositol turnover, by the low levels of 1,2-diacylglycerol generated by GCPs, and by the resistance of AA release to treatment of GCPs with RHC-80267, a specific inhibitor of diacylglycerol lipase. The predominance of PLA₂ activity in GCPs raises questions regarding its regulation and the functional roles of PI metabolites, especially lysocompounds, in growth cones.     

Effects of Fusaric Acid on Reactive Oxygen Species and Antioxidants in Tomato Cell Cultures

Generation of O₂^– and H₂O₂ as well as the activities of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, dehydroascorbate reductase and ascorbate content were studied in tomato cell cultures in response to fusaric acid – a nonspecific toxin of phytopathogenic Fusarium species. Toxin treatment resulted in decreased cell viability which was preceded by culture medium alkalinization up to 0.65 pH unit and enhanced extracellular O₂^– production. The H₂O₂ level was not significantly affected. In toxin-treated cultures, a transient, significant increase occurred in intracellular superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase activities. Fusaric acid-induced ascorbate turnover modulation led to up to a twofold increase in dehydroascorbic acid accumulation, and a decrease in the associated ascorbate redox ratio. It was concomitant with a significant decrease in dehydroascorbate reductase activity. These results support previous observations that the pro- and anti-oxidant systems are involved in response to fusaric acid treatment although differential response of H₂O₂ and its metabolism-related enzymes between the whole leaf and cell culture assays was found.     

Some kinetic properties of polyphenol oxidase from Thymbra spicata L. var. spicata

Polyphenol oxidase (PPO) of Thymbra (Thymbra spicata L. var. spicata) was isolated by (NH₄)₂SO₄ precipitation and dialysis. A diphenolase from Thymbra plant, active against 4-methylcatechol, catechol and pyrogallol was characterized in detail in terms of pH and temperature optima, stability, kinetic parameters and inhibition behaviour towards some general PPO inhibitors. 4-Methylcatechol was the most suitable substrate, due to the lowest K_m and the biggest V_max/K_m values, followed by catechol and pyrogallol. The Thymbra PPO had maximum activity at pH 5.0, 7.0 and 8.0 with 4-methylcatechol, catechol and pyrogallol substrates, respectively. The optimum temperature of activity for Thymbra PPO was 30, 40 and 50 °C for 4-methylcatechol, catechol and pyrogallol substrates, respectively. It was found that optimum temperature and pH were substrate-dependent studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time. Inhibition of Thymbra PPO was investigated with inhibitors such as l-cysteine and glutathione using 4-methylcatechol, catechol and pyrogallol as substrates. It was found that l-cysteine was a more effective inhibitor than glutathione owing to lower K_i. The type of inhibition depended on the origin of the PPO studied and also on the substrate used. Furthermore, the IC₅₀ values of inhibitors sudied on PPO were determined by means of activity percentage (I) diagrams.     

Improved methods for the extraction of polyphenol oxidase from d'anjou pears

The conditions for extracting polyphenol oxidase (PPO, monophenol monooxygenase, EC 1.14.18.1) from d'Anjou pears have been studied. Water extracts of pear PPO contained artefacts which were present as additional bands on polyacrylamide-gel electrophoresis. Buffer extracts of an acetone powder did not remove sufficient endogenous phenolics to prevent browning of the extract. The following phenolic absorbents, arranged in order of increasing efficiency, reduced the formation of artefacts in extracts of PPO: PVPP, Amberlite XAD-4, Bio-Rad AG 1-X8, and Bio-Rad AG 2-X8. Greatest activity was extracted within a pH range of 5.6–5.9. Anion exchange resins were particularly effective in removing phenolics. XAD-4, AG 1-X8, or AG 2-X8 did not adsorb PPO and reduced the electrophoretically separable bands of PPO activity from 11 in water extracts to 3. The properties of the crude PPO were also studied.     

Studies on the lactoperoxidase system: reaction kinetics and antibacterial activity using two methods for hydrogen peroxide generation

D.A. DIONYSIUS, P.A. GRIEVE AND A.C. VOS. 1992. Components of the lactoperoxidase system were measured during incubation in Isosensitest broth, with enzymatic (glucose oxidase, GO) or chemical (sodium carbonate peroxyhydrate, SCP) means to generate H₂O₂. When low levels of thiocyanate (SCN^-) were used in the GO system, H₂O₂ was detected and lactoperoxidase (LP) was inactivated when SCN^- was depleted. With 10-fold higher SCN^-, LP remained active and H₂O₂ was not detectable. The oxidation product of the LP reaction, most likely hypothiocyanite, was present in low concentrations. When SCP was used for the immediate generation of H₂O₂ in a system employing low SCN^-, half the LP activity was lost within minutes but thereafter it remained stable. Low concentrations of oxidation product were measured and H₂O₂ was not detected during the course of the experiment. At high SCN^- levels, relatively high concentrations of oxidation product were produced immediately, with H₂O₂ undetectable. The results suggest that the final product of the LP reaction depends on the method of H₂O₂ generation and the relative proportions of the substrates. Antibacterial activity of the two LPS was tested against an enterotoxigenic strain of Escherichia coli. Both systems showed bactericidal activity within 4 h incubation at 37°C.     

Detection of hydrogen peroxide produced by meat lactic starter cultures

Twelve strains of meat lactic starter cultures (Pediococcus spp. and Lactobacillus plantarum) were found to produce hydrogen peroxide in vitro. The (cumulative) amounts of H₂O₂ produced were measured through the peroxidative action of catalase on H₂O₂ and oxidation of added formate to CO₂ by the H₂O₂-catalase complex formed. There was a problem in building a calibration curve for converting values of formate oxidation into amounts of H₂O₂, either by adding H₂O₂ directly to the assay mixture or having it produced via a glucose-glucose oxidase system.