Enhanced Peroxidase Activity Research Articles

Activation of systemic disease resistance in pea by an avirulent bacterium or a benzothiadiazole, but not by a fungal leaf spot pathogen

Inoculation of first expanded leaves of pea seedlings with an avirulent strain of Pseudomonas syringae pv. pisi, or treatment with sprays of a benzothiadiazole (20 or 100 μg a.i. mL−1), decreased the susceptibility of subsequent leaves 7 or 14 days later to challenge inoculation with Mycosphaerella pinodes. Inoculation of first leaves with a virulent strain of P. syringae pv. pisi or with M. pinodes did not decrease the susceptibility of plants to M. pinodes. Treatments effective in decreasing susceptibility to M. pinodes were similarly active against Uromyces viciae‐fabae and virulent P. syringae pv. pisi. Effective treatments also enhanced the activities of the enzymes β‐1,3‐glucanase and chitinase in untreated upper leaves 6 days later. Ineffective treatments for decreased susceptibility had no effect on the activity of the enzymes. None of the treatments enhanced peroxidase activities. The results are discussed in relation to the reported signalling effects of the benzothiadiazole and in relation to a suggested high activity of the avirulent P. syringae pv. pisi strain and inactivity of M. pinodes in enhancing natural signalling.

Haematoporphyrin enhances the peroxidase activity of haemoglobin

The effect of haematoporphyrin, a component of some of the widely used anticancer drugs, on the peroxidase activity of haemoglobin has been studied. Haematoporphyrin increases the haemoglobin-catalysed hydrogen peroxide-mediated oxidation of o-dianisidine or NADH. Spectrophotometric study reveals that an interaction occurs between haemoglobin and haematoporphyrin which leads to a conformational change of the protein. The extent of enhanced peroxidase activity as well as conformational change of the protein vary in a positive manner with the stoichiometric ratio of haematoporphyrin/haemoglobin. An increase in the peroxidase activity of haemoglobin was also observed in the presence of superoxide dismutase, which catalysed the removal of superoxide anion generated during autoxidation of haemoglobin. Possible mechanisms underlying the relation between the conformational change of haemoglobin due to its interaction with haematoporphyrin and the enhanced peroxidase activity are discussed.

Azo dye-mediated regulation of total phenolics and peroxidase activity in thyme (Thymus vulgaris L.) and rosemary (Rosmarinus officinalis L.) clonal lines.

Thyme (Thymus vulgaris L.) and rosemary (Rosmarinus officinalis L.) clonal lines, which were previously isolated from a heterogeneous seed population by plant tissue culture techniques, have been targeted as potential plants for phytoremediation of organic pollutants such as azo dyes and related aromatic compounds. Three thyme clonal lines and three rosemary clonal lines were tested for the ability to grow on hormone-free medium containing 0.01% of azo dye Poly S-119. The results showed that dye tolerance was associated with reduced phenolics and enhanced peroxidase activity in these clonal lines. There was a clear inverse correlation between total phenolics and peroxidase activity in these plants in response to Poly S-119. The tolerance of these clonal lines showed variations at different growing stages. These observations suggested that the peroxidase activity was inducible. Because peroxidases are involved in lignification, wound healing, aromatic compound degradation, pathogen defense, and stiffening, the results suggest that azo dye stimulated the defense response of thyme and rosemary clonal plants by increasing the peroxidase activity. Stereomicroscopic observations revealed that the azo dye was sequestered within the growing axis of the plant roots, which may also enhance the polymerization of azo dye onto the cell wall with the help of enhanced peroxidase activity.

Nitric Oxide Modulates the Catalytic Activity of Myeloperoxidase

Myeloperoxidase (MPO), an abundant protein in neutrophils, monocytes, and subpopulations of tissue macrophages, is believed to play a critical role in host defenses and inflammatory tissue injury. To perform these functions, an array of diffusible radicals and reactive oxidant species may be formed through oxidation reactions catalyzed at the heme center of the enzyme. Myeloperoxidase and inducible nitric-oxide synthase are both stored in and secreted from the primary granules of activated leukocytes, and nitric oxide (nitrogen monoxide; NO) reacts with the iron center of hemeproteins at near diffusion-controlled rates. We now demonstrate that NO modulates the catalytic activity of MPO through distinct mechanisms. NO binds to both ferric (Fe(III), the catalytically active species) and ferrous (Fe(II)) forms of MPO, generating stable low-spin six-coordinate complexes, MPO-Fe(III).NO and MPO-Fe(II).NO, respectively. These nitrosyl complexes were spectrally distinguishable by their Soret absorbance peak and visible spectra. Stopped-flow kinetic analyses indicated that NO binds reversibly to both Fe(III) and Fe(II) forms of MPO through simple one-step mechanisms. The association rate constant for NO binding to MPO-Fe(III) was comparable to that observed with other hemoproteins whose activities are thought to be modulated by NO in vivo. In stark contrast, the association rate constant for NO binding to the reduced form of MPO, MPO-Fe(II), was over an order of magnitude slower. Similarly, a 2-fold decrease was observed in the NO dissociation rate constant of the reduced versus native form of MPO. The lower NO association and dissociation rates observed suggest a remarkable conformational change that alters the affinity and accessibility of NO to the distal heme pocket of the enzyme following heme reduction. Incubation of NO with the active species of MPO (Fe(III) form) influenced peroxidase catalytic activity by dual mechanisms. Low levels of NO enhanced peroxidase activity through an effect on the rate-limiting step in catalysis, reduction of Compound II to the ground-state Fe(III) form. In contrast, higher levels of NO inhibited MPO catalysis through formation of the nitrosyl complex MPO-Fe(III)-NO. NO interaction with MPO may thus serve as a novel mechanism for modulating peroxidase catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.

Loss of oxidation-reduction specificity in amyotrophic lateral sclerosis-associated CuZnSOD mutants.

Both transgenic mouse and cell culture models of familial amyotrophic lateral sclerosis (FALS) support a gain-of-function effect for the mutations in copper-zinc superoxide dismutase (CuZnSOD) associated with FALS, but the nature of the function gained remains incompletely characterized. We previously reported an enhanced peroxidase activity for FALS-associated CuZnSOD mutants. Because one of the targets of such activity is CuZnSOD itself, we examined peroxide-mediated inactivation of wild-type and mutant CuZnSODs, and found that the mutants are more readily inactivated. Inactivation of the mutants was associated with fragmentation, which did not occur in the wild-type enzyme under these conditions. Furthermore, the reduction of the FALS-associated mutants by ascorbate was enhanced markedly when compared to the wild-type enzyme. The visible spectra of the mutants showed a consistent blue shift of the peak at 680 nm in the wild-type enzyme, suggesting an alteration in copper-site geometry. These results extend previous studies demonstrating enhanced peroxidase activity in the mutants, and suggest that the toxic function that leads to motor neuron degeneration may result from a loss of specificity of the redox reactions catalyzed by CuZnSOD.

Superoxide dismutase-1 mutation-related neurotoxicity in familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neuron system involvement, and is epidemiologically subclassified into sporadic, familial and endemic forms. About 20% of ALS families are associated with mutations in the gene for superoxide dismutase-1 (SOD1) encoded on chromosome 21q22.1. Several studies have pointed to a variety of functions of mutant SOD1, which has enhanced catalytic activity of the peroxynitrite-mediated tyrosine nitration, readily releases the reactive Cu ions, induces apoptotic cell death, has enhanced peroxidase activity, damages the mitochondria to release Ca2+, and forms SOD1-containing aggregates in the cytoplasm. Many of these studies have obtained evidence for increased oxidative damage in ALS. On the other hand, some reports disagree with oxidative damage involvement in SOD1 mutant ALS. In considering the findings of increased oxidative damage in mutant SOD1-expressing transgenic mice, it should be remembered that overexpression of mutant SOD1 may enhance oxidative stress generation from this enzyme. In this review, we present the clinicopathological features of SOD1 mutant familial ALS and its transgenic mouse model, and also discuss SOD1 mutation-related neurotoxicity, including SOD1 protein aggregation and post-translational protein modification.

Surfactant-histidine-heme ternary complex as a simple artificial heme enzyme in organic media

A surfactant-heme complex which shows peroxidase activity in organic media has been prepared by a method utilizing water-in-oil (W/O) emulsions. Both the aqueous phase pH and the type of surfactant appeared to have prominent effect on the catalytic activity of the heme complex in benzene. The catalytic efficiency of the heme complex was enhanced more than ten times by adding histidine to the aqueous phase of W/O emulsions in the preparation process. The enhancement of peroxidase activity was observed only in a nonaqueous medium due to the increase of the effective concentration of histidine as an activator. In the present study, we propose a simple preparation method for an artificial heme enzyme which works in nonaqueous media. Copyright 1999 John Wiley & Sons, Inc.

Nitric oxide modulates the catalytic activity of myeloperoxidase

Myeloperoxidase (MPO), an abundant protein in neutrophils, monocytes, and subpopulations of tissue macrophages, is believed to play a critical role in host defenses and inflammatory tissue injury. To perform these functions, an array of diffusible radicals and reactive oxidant species may be formed through oxidation reactions catalyzed at the heme center of the enzyme. Myeloperoxidase and inducible nitric-oxide synthase are both stored in and secreted from the primary granules of activated leukocytes, and nitric oxide (nitrogen monoxide; NO) reacts with the iron center of hemeproteins at near diffusion-controlled rates. We now demonstrate that NO modulates the catalytic activity of MPO through distinct mechanisms. NO binds to both ferric (Fe(III), the catalytically active species) and ferrous (Fe(II)) forms of MPO, generating stable low-spin six-coordinate complexes, MPO-Fe(III)·NO and MPO-Fe(II)·NO, respectively. These nitrosyl complexes were spectrally distinguishable by their Soret absorbance peak and visible spectra. Stopped-flow kinetic analyses indicated that NO binds reversibly to both Fe(III) and Fe(II) forms of MPO through simple one-step mechanisms. The association rate constant for NO binding to MPO-Fe(III) was comparable to that observed with other hemoproteins whose activities are thought to be modulated by NO in vivo. In stark contrast, the association rate constant for NO binding to the reduced form of MPO, MPO-Fe(II), was over an order of magnitude slower. Similarly, a 2-fold decrease was observed in the NO dissociation rate constant of the reducedversus native form of MPO. The lower NO association and dissociation rates observed suggest a remarkable conformational change that alters the affinity and accessibility of NO to the distal heme pocket of the enzyme following heme reduction. Incubation of NO with the active species of MPO (Fe(III) form) influenced peroxidase catalytic activity by dual mechanisms. Low levels of NO enhanced peroxidase activity through an effect on the rate-limiting step in catalysis, reduction of Compound II to the ground-state Fe(III) form. In contrast, higher levels of NO inhibited MPO catalysis through formation of the nitrosyl complex MPO-Fe(III)-NO. NO interaction with MPO may thus serve as a novel mechanism for modulating peroxidase catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.

Phytohormone regulation of isoperoxidases in catharanthus roseus suspension cultures

Peroxidase (POD) activity was investigated in Catharanthus roseus cell suspensions cultured under different hormonal conditions. Depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) from the culture medium enhanced POD activity in cells and spent medium. Addition of phytohormones, in particular the auxin 2,4-D, reduced POD activity in medium and cellular compartments and enhanced ionically cell-wall bound POD. The differential modulation of POD is due to hormone effects on synthesis and⧹or accumulation of POD, rather than on the secretion process. Qualitative analysis showed that 2,4-D, but not cytokinins, regulated the synthesis of a basic isoform. The cytokinin treatment seemed to affect acidic rather than basic isoforms. The presence of basic POD is correlated with the capacity of cells to produce indole alkaloids. The major extracellular basic isoperoxidase was purified to homogeneity from culture medium of Catharanthus roseus cell suspensions. The isolated peroxidase is a haem protein with a M r of 33,000 and a pI close to 9. The effect of pH on peroxidase activity was studied using guaiacol as substrate and the optimum pH determined at 25° was 6.0. This enzyme acted on guaiacol, 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-dianisidine, o-phenylenediamine ( o-PD) and pyrogallol, but had no effect on syringaldazine or coniferyl alcohol substrates.

In vitro evolution of horse heart myoglobin to increase peroxidase activity.

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k1 (for H2O2 oxidation of metmyoglobin) of 1. 34 x 10(4) M-1 s-1 ( approximately 25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2'-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 x 10(6) M-1 s-1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1 degreesC vs. 76.5 degreesC). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 x 10(-6) M-1 s-1 vs. 22.3 x 10(-6) M-1 s-1; koff, 24.3 s-1 vs. 24.2 s-1; KO2, 0.91 x 10(-6) M-1 vs. 0.92 x 10(-6) M-1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 x 10(-6) M-1s-1 vs. 0.51 x 10(-6) M-1s-1; koff, 5.08 s-1 vs. 3.51 s-1; KCO, 1.77 x 10(-7) M-1 vs. 1.45 x 10(-7) M-1). All four substitutions are in the heme pocket and within 5 A of the heme group.

Atrazine effects on estuarine macrophytes Spartina alterniflora and Juncus roemerianus

AbstractTwo dominant estuarine marsh plants, Juncus roemerianus and Spartina alterniflora, were exposed for 35 d to three dose levels of atrazine with measured concentrations of approximately 0.03, 0.25, and 3 mg/L to assess peroxidase (POD) activity, lipid peroxidation, chlorophyll changes, and growth rates. The objective of the study was to evaluate toxicity of atrazine and to measure multiple endpoints to assess their value as biomarkers of atrazine stress or resistance. In S. alterniflora, exposure to 3.1 mg/L of atrazine did not significantly inhibit growth, generated no lipid peroxidation products, and did not affect chlorophyll production, but did significantly enhance POD activity. In contrast, in J. roemerianus, atrazine exposure significantly inhibited growth at 3.8 mg/L, produced lipid peroxidation products and inhibited chlorophyll production in a dose‐response fashion, and did not elicit a POD response. In combination, POD activity and lipid peroxidation are useful indicators of atrazine stress to these plants. Results of the study indicate that J. roemerianus would decline or die off if exposed to chronically high environmental concentrations of atrazine, whereas minimal damage would occur to S. alterniflora.

Familial amyotrophic lateral sclerosis and Cu/Zn superoxide dismutase mutation

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that involves mainly the motor neuron system. Five to 10 percent of the ALS cases are familial; most others are sporadic. Several mutations in the superoxide dismutase‐1 (SOD1) gene have recently been shown to be associated with about 20% of familial ALS patients. The reduced enzyme activity of many mutant SOD1 points to the possibility that a loss‐of‐function effect of the mutant enzyme is responsible for the pathogenesis of the disease. However, this conflicts with the autosomal dominant inheritance of SOD1 mutation‐associated ALS and the normal SOD1 activity in homozygous patients in a SOD1‐linked ALS family. Current biochemical investigations have provided evidence that mutant SOD1 may catalyze the peroxynitrite‐mediated nitration of protein tyrosine residues, release copper and zinc ions, facilitate apoptosis of neurons and have enhanced peroxidase activity. Immunocytochemical studies demonstrated the presence of intense SOD1 immunoreactivity in Lewy body‐like inclusions, which are characteristic features of a certain form of familial ALS with posterior column involvement, in the lower motor neurons of patients in ALS families with different SOD1 mutations. More recently, strains of transgenic mice expressing mutant SOD1 have been established. These mice clinicopathologically develop a motor neuron disease mimicking human ALS with the exception of pronounced intraneuronal vacuolar degeneration. The overexpression of wild‐type SOD1 in mice has failed to give rise to the disease. Only one transgene for mutant SOD1 is enough to cause motor neuron degeneration and the severity of clinical course correlates with the transgene copy number. These observations in SOD1‐linked familial ALS and its transgenic mouse model suggest a novel neurotoxic function of mutant SOD1.

Elicitor-induced extensin insolubilization in suspension-cultured tomato cells

Insolubilization of cell wall hydroxyproline-rich glycoproteins is a common plant defence response when plants are challenged with either pathogens or elicitors derived from pathogens. A yeast elicitor that insolubilized cell wall extensin of suspension-cultured tomato cells was purified by 80% ethanol precipitation of yeast extract followed by anion-exchange chromatography, gel-filtration chromatography and reverse phase HPLC. MALDI/TOF-mass spectrum analysis of the purified elicitor preparation generated one major peak at 410 ± Da. Elicitor activity was inactivated at 100° for 10 min or after incubation with pronase. No loss of elicitor activity was observed after periodate treatment. Polyclonal antiserum raised against native tomato extensin was used to demonstrate extensin insolubilization in vivo. Salt-elutability of extensin from tomato cells was dependent upon concentration and time of incubation of the cells with elicitor. SDS-PAGE/western blotting of salt-eluted protein from elicited cells also demonstrated insolubilization of HRGPs in the cell wall. The elicitor was found to stimulate cell wall peroxidase activity and extensin insolubilization in isolated tomato cell walls and in intact cells. The elicitor also induced a transient oxidative burst which began after 5 min and was maximal after 20 min incubation with the cells. Other typical plant defence responses known to be triggered by elicitors, such as changes in H +, K + or Ca 2+ fluxes, enhanced activities of lipoxygenase, phenylalanine ammonia lyase and superoxide dismutase, were not observed when tomato cells were challenged with this elicitor. The Ca 2+ channel blocker verapamil did not prevent elicitor-induced extensin insolubilization in whole cells. This study suggests that biochemical events, such as transient oxidative burst and enhanced peroxidase activity, which accompany extensin insolubilization involve the cell wall without any apparent plasma membrane participation. The above cell wall events may be sufficient to form the early plant defence responses to microbial challenge. © 1997 Elsevier Science Ltd. All rights reserved

Enhancement of peroxidase activity by stress-related chemicals in sweet potato

Stress-related plant hormones, abscisic acid (ABA, 0.5 mg l −1) or ethephon (0.5 ml l −1, an ethylene generating compound), enhanced peroxidase (POD) activity by ca 50% in suspension cultures of sweet potato ( Ipomoea batatas), depending on the time after treatment when added to medium at the late logarithmic growth phase. Calcium chloride (50 mM) also increased POD activity by ca 20%, whereas paraquat ( 1 mg l −1) slightly stimulated it soon after treatment, but inhibited it after 13 days. ABA was the most effective in causing increased POD activity, i.e. 11 247 units g −1 dry cell wt (substrate, pyrogallol) 18 days after treatment, which was 1.78 times higher than a normal culture. However, the four chemicals used had no effect on the level of extracellular POD, which was maintained at 5–10% of total POD activity regardless of chemical treatment.

Peroxidase changes in barley induced by ionizing and thermal radiation

Thermal and ionizing (gamma-ray) radiations were used to induce damage to barley seeds (IB65). The activity and isozyme banding patterns of peroxidase were compared. It was found that both physical agents caused damage to barley seeds (as observed from seedling height), but their action on peroxidase activity is not similar. Gamma-Rays enhance peroxidase activity. Thermal radiation, on the other hand, tends to reduce it but fails to alter the number of peroxidase isozymes. It is conjectured that the pathways of damage by thermal and ionizing radiations are not the same.

Peroxidase Activity of Cytochrome c Generated by Phenylboronic Acid Modification

Abstract Reactive amino residues of horse heart cytochrome c were successfully modified with 4-boronobenzoic acid N-hydroxysuccinimide ester 1. The modified cytochrome c shows an enhanced peroxidase activity, which can be regulated by the addition of sugars.

The association between heat-induced susceptibility of tomato to Meloidogyne incognita and peroxidase activity

Resistant tomato seedlings (cultivar VFN8) were tested for their reaction to the root-knot nematode Meloidogyne incognita at five temperatures between 27 and 38°C. The data obtained showed that the seedlings became increasingly susceptible to M. incognita above 30°C with an optimum at 34°C. Seedlings incubated and inoculated at 34°C, after 2 days showed a susceptible response, i.e., reduced necrosis and a higher number of galls. A temporary break of resistance could also be induced by conditioning uninoculated seedlings at higher temperatures for 6 days. The plants remained susceptible for 1-2 days when the temperature was shifted from 34 to 27°C before inoculation. Chromatographic and electrophoretic analyses demonstrated that enhanced peroxidase activity accompanied nematode infection at 27°C. High-temperature-induced susceptibility of tomato to M. incognita was correlated with low to no increase in peroxidase activity. The increase of peroxidases at 27°C was due to an increase in activity of constitutive enzymes. Heat treatment reduced the lignin level of both infected and uninfected roots, indicating that the process of lignification was partially blocked by high temperature. The data provide evidence that heat-induced susceptibility is associated with a reduced hypersensitive reaction, and that there is a correlation between hypersensitivity and reduced peroxidase and lignin levels.

Enhancement of Peroxidase Activity in the Apoplastic Matrix of Cucurbita Pepo l. Plants Exposed to O3

(1995). Enhancement of Peroxidase Activity in the Apoplastic Matrix of Cucurbita Pepo l. Plants Exposed to O3. Giornale botanico italiano: Vol. 129, No. 4, pp. 1139-1140.

Enhanced Peroxidase Activity of Cytochrome c by Phosphate Bilayer Membrane

Abstract Peroxidase activity of cytochrome c (cyt-c) as measured by oxidation of o-methoxyphenol with H2O2, was enhanced due to binding of cyt-c to a phosphate bilayer membrane. The rate enhancement and the membrane binding were not observed with ammonium and zwitterionic bilayer membranes.

Peroxidase as a developmental marker in plant tissue culture.

Peroxidase was studied as a developmental marker in pumpkin (Cucurbita pepo L.) callus lines and horse-radish (Armoracia lapathifolia Gilib) transformants. Embryogenic callus lines DE grown on MS medium with 2.4-D and NA-3 grown on medium with NAA and adenine sulfate showed about a 20 times higher enzyme activity than the habituated non-embryogenic line Z5b/T grown on medium without hormones. A rise in peroxidase activity indicated that somatic embryogenesis was triggered in a few habituated tissue cultures. Separated globular embryoids had a manifold lower enzyme activity than the callus from which they originated. SDS-electrophoresis showed distinct polypeptide patterns between the horse-radish leaves and crown galls, but the tumor characteristic protein bands failed to be identified. In horse-radish crown galls and short bushy plants regenerated from hairy roots an enhanced peroxidase activity was registered. Due to its high peroxidase level and abundant biomass production horse-radish transformants should facilitate enzyme production.