Activation of ascorbate-glutathione cycle inArabidopsis leaves in response to aminotriazole

Aminotriazole(AT)-induced changes in growth, hydrogen peroxide content and activities of H₂O₂-scavenging antioxidant enzymes were investigated in the growing leaves ofArabidopsis plants (Arabidopsis thaliana cv Columbia). Catalase activity of rosette leaves was reduced by 65% with an application of 0.1 mM AT (a herbicide known as a catalase inhibitor), whereas the leaf growth and H₂O₂ content were almost unaffected. However, an approximate 1.6 to 2-fold increase in cytosolic ascorbate peroxidase (APX) activity concomitant with a substantial activation of glutathione reductase (GR) (approx. 22% increase) was observed during leaf growth in the presence of 0.1 mM AT. The activity of cytosolic APX in leaves was also increased by 1.8-fold with an application of exogenous 2 mM paraquat (an inducer of H₂O₂ production in plant cells) in the absence of AT. These results collectively suggest that (a) cytosolic APX and GR operate to activate an ascorbate-glutathione cycle for the removal of H₂O₂ under severe catalase deactivation, and (b) the expression of APX seems to be regulated by a change of the endogenous H₂O₂ level in leaf cells.     

The mechanism of stomatal closing by salicylic acid inCommelina communis L.

The mechanism of stomatal closing by salicylic acid (SA) has been investigated. The addition of 1 mM SA to fully opened stomata resulted in a significant reduction of 75% in stomatal aperture. Stomata in the treatment of SA with EGTA closed as observed in the treatment of SA. However, the addition of catalase with SA completely inhibited stomatal closing. Stomatal closing induced by SA was also reduced by Ca²⁺. To understand the relation bewteen stomatal closing by SA and catalase activity, the effect of SA on catalse activity and the effect of AT (catalase inhibitor) on stomatal closing was investigated. SA inhibited 32% of catalase activity. Stomata in isolated epidermis floated on an incubation solution containing 0.1 mM AT closed from 9.6 μm to 3.2 μm after 1 hour. SA stimulated K⁺ efflux as much as the twice of the control in isolated strips. SA inhibited 53% of photosynthetic activity at the light intensity of 1000 μmole m² s¹ on SA infiltrated leaves. A similar result was found on stomatal conductance in SA infiltrated leaves. These results indicate that SA inhibit catalase activity and increase the concentration of H₂O₂ in guard cell cytoplasm. H₂O₂ oxidize the plasma membrane and increase the membrane permeability of K⁺. The mass efflux of K⁺ induce the loss of turgor pressure and lead to stomatal closing. The inhibition of photosynthetic activity by SA suggests that stomatal closing by SA is also related with the decrease of photosynthetic activity.     

Response of Plant-Colonizing Pseudomonads to Hydrogen Peroxide

Colonization of plant root surfaces by Pseudomonas putida may require mechanisms that protect this bacterium against superoxide anion and hydrogen peroxide produced by the root. Catalase and superoxide dismutase may be important in this bacterial defense system. Stationary-phase cells of P. putida were not killed by hydrogen peroxide (H₂O₂) at concentrations up to 10 mM, and extracts from these cells possessed three isozymic bands (A, B, and C) of catalase activity in native polyacrylamide gel electrophoresis. Logarithmic-phase cells exposed directly to hydrogen peroxide concentrations above 1 mM were killed. Extracts of logarithmic-phase cells displayed only band A catalase activity. Protection against 5 mM H₂O₂ was apparent after previous exposure of the logarithmic-phase cells to nonlethal concentrations (30 to 300 μM) of H₂O₂. Extracts of these protected cells possessed enhanced catalase activity of band A and small amounts of bands B and C. A single form of superoxide dismutase and isoforms of catalase were apparent in extracts from a foliar intercellular pathogen, Pseudomonas syringae pv. phaseolicola. The mobilities of these P. syringae enzymes were distinct from those of enzymes in P. putida extracts.     

Oxygen consumption by purified plasmalemma vesicles from wheat roots: Stimulation by NADH and salicylhydroxamic acid (SHAM)

Plasmalemma vesicles from wheat (Triticum aestivum L.) roots consumed O₂ and the addition of 1 mM NADH increased the rate ~ 3-fold (to 15-30 nmol O₂·mg⁻¹·min⁻¹). The NADH-dependent O₂ uptake was abolished by catalase. In the presence of salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase pathway in plant mitochondria, NADH-dependent O₂ consumption was stimulated 10–20-fold (to 200–400 nmol·mg^1̄·min⁻¹). Catalase also abolished this stimulation, which was KCN-sensitive but antimycin A-insensitive, and the production of H₂O₂ during SHAM-stimulated NADH-dependent O₂ uptake was demonstrated. Irrespective of the mechanism, SHAM-stimulated respiration by root plasmalemma makes it difficult to interpret results on root respiration obtained using KCN and SHAM.     

Heterologous extracellular expression and initial characterization of the peroxisomal catalase from the methylotrophic yeast Hansenula polymorpha in Pichia pastoris

Catalase is well known to eliminate H₂O₂ in cells and reduces the toxicity of peroxide compounds. A catalase gene HpCAT1 of methylotrophic yeast Hansenula polymorpha without the part coding the native signal peptide was cloned into expression vector pYM3165 and then integrated into genome of Pichia pastoris GS115 by electroporation. The result of the enzyme activity assay and SDS-PAGE demonstrated that the recombinant protein (HpCAT1) of H. polymorpha was extracellularly expressed in P. pastoris. The expressed catalase was recovered from the culture supernatant of P. pastoris GS115 and purified by (NH₄)₂SO₄ fractionation and Ni-NTA affinity chromatography. The main biochemical properties of the recombinant protein HpCAT1, such as thermodependence and thermostability, pH optimum and pH stability, as well as the effect of metal ions and chemicals, were characterized. With H₂O₂ as the substrate, HpCAT1 displayed pH and temperature optima of ～2.6 and 45°C, respectively. The recombinant HpCAT1 activity was inhibited by 1 mM Hg²⁺ and Cu²⁺, but was highly enhanced by 1.0 mM Fe²⁺.     

A Mechanism of Resistance to Hydrogen Peroxide in Vibrio rumoiensis S-1

A possible mechanism of resistance to hydrogen peroxide (H₂O₂) in Vibrio rumoiensis, isolated from the H₂O₂-rich drain pool of a fish processing plant, was examined. When V. rumoiensis cells were inoculated into medium containing either 5 mM or no H₂O₂, they grew in similar manners. A spontaneous mutant strain, S-4, derived from V. rumoiensis and lacking catalase activity did not grow at all in the presence of 5 mM H₂O₂. These results suggest that catalase is inevitably involved in the resistance and survival of V. rumoiensis in the presence of H₂O₂. Catalase activity was constitutively present in V. rumoiensis cells grown in the absence of H₂O₂, and its occurrence was dependent on the age of the cells, a characteristic which is observed for the HP II-type catalase of Escherichia coli. The presence of the HP II-type catalase in V. rumoiensis cells was evidenced by partial sequencing of the gene encoding the HP II-type catalase from this organism. A notable difference between V. rumoiensis and E. coli is that catalase is accumulated at very high levels (~2% of the total soluble proteins) in V. rumoiensis, in contrast to the case for E. coli. When V. rumoiensis cells which had been exposed to 5 mM H₂O₂ were centrifuged, most intracellular proteins, including catalase, were recovered in the medium. On the other hand, when V. rumoiensis cells were grown on plates containing various concentrations of H₂O₂, individual cells had a colony-forming ability inferior to those of E. coli, Bacillus subtilis, and Vibrio parahaemolyticus. Thus, it is suggested that when V. rumoiensis cells are exposed to high concentrations of H₂O₂, most cells will immediately be broken by H₂O₂. In addition, the cells which have had little or no damage will start to grow in a medium where almost all H₂O₂ has been decomposed by the catalase released from broken cells.     

Hydrogen peroxide metabolism and sensing in human erythrocytes: A validated kinetic model and reappraisal of the role of peroxiredoxin II

Hydrogen peroxide (H₂O₂) metabolism in human erythrocytes has been thoroughly investigated, but unclear points persist. By integrating the available data into a mathematical model that accurately represents the current understanding and comparing computational predictions to observations we sought to (a) identify inconsistencies in present knowledge, (b) propose resolutions, and (c) examine their functional implications. The systematic confrontation of computational predictions with experimental observations of the responses of intact erythrocytes highlighted the following important discrepancy. The high rate constant (10⁷–10⁸ M⁻¹ s⁻¹) for H₂O₂ reduction determined for purified peroxiredoxin II (Prx2) and the high abundance of this protein indicate that under physiological conditions it consumes practically all the H₂O₂. However, this is inconsistent with extensive evidence that Prx2’s contribution to H₂O₂ elimination is comparable to that of catalase. Models modified such that Prx2’s effective peroxidase activity is just 10⁵ M⁻¹ s⁻¹ agree near quantitatively with extensive experimental observations. This low effective activity is probably due to a strong but readily reversible inhibition of Prx2’s peroxidatic activity in intact cells, implying that the main role of Prx2 in human erythrocytes is not to eliminate peroxide substrates. Simulations of the responses to physiological H₂O₂ stimuli highlight that a design combining abundant Prx2 with a low effective peroxidase activity spares NADPH while improving potential signaling properties of the Prx2/thioredoxin/thioredoxin reductase system.     

Peroxynitrite and hydrogen peroxide elicit similar cellular stress responses mediated by the Ccp1 sensor protein

Peroxynitrite [ONOO(H)] is an oxidant associated with deleterious effects in cells. Because it is an inorganic peroxide that reacts rapidly with peroxidases, we speculated that cells may respond to ONOO(H) and H₂O₂ challenge in a similar manner. We exposed yeast cells to SIN-1, a well-characterized ONOO(H) generator, and observed stimulation of catalase and peroxiredoxin (Prx) activities. Previously, we reported that H₂O₂ challenge increases these activities in wild-type cells and in cells producing the hyperactive mutant H₂O₂ sensor Ccp1^W191F but not in Ccp1-knockout cells (ccp1Δ). We find here that the response of ccp1Δ and ccp1^W191F cells to SIN-1 mirrors that to H₂O₂, identifying Ccp1 as a sensor of both peroxides. SIN-1 simultaneously releases ^•NO and O₂^•−, which react to form ONOO(H), but exposure of the three strains separately to an ^•NO donor (spermine-NONOate) or an O₂^•− generator (paraquat) mainly depresses catalase or Prx activity, whereas co-challenge with the NONOate and paraquat stimulates these activities. Because Ccp1 appears to sense ONOO(H) in cells, we examined its reaction with ONOO(H) in vitro and found that peroxynitrous acid (ONOOH) rapidly (k₂>10⁶ M⁻¹ s⁻¹) oxidizes purified Ccp1 to an intermediate with spectral and ferrocytochrome-oxidizing properties indistinguishable from those of its well-characterized compound I formed with H₂O₂. Importantly, the nitrite released from ONOOH is not oxidized to ^•NO₂ by Ccp1^׳s compound I, unlike peroxidases involved in immune defense. Overall, our results reveal that yeast cells mount a common antioxidant response to ONOO(H) and H₂O₂, with Ccp1 playing a pivotal role as an inorganic peroxide sensor.     

Partial purification and characterization of peroxidases from the leaves of Sapindus mukorossi

Peroxidases were isolated from Sapindus mukorossi (Reetha) and partially purified using acetone precipitation, ion-exchange chromatography with a 14-fold purification, 22% recovery and a specific activity of 266?×?10³ units/mg protein. Sapindus peroxidases (SPases) showed six bands after acetone precipitation and one distinct band after ion exchange chromatography on Native-PAGE after zymography. Enzymes purified by ion exchange chromatography were loaded on Sepahdex G-50 superfine column and their molecular weight was reported to be 25?kDa. They showed temperature optima at 50°C and pH optima at 5.0.?km for SPases was reported to be 1.05?mM and 0.186?mM for guaiacol and H₂O₂ respectively. The V_max/K_m value for o-dianisidine was 449 while for H₂O₂ it was 5?×?10⁵. Protocatechuic acid acts as a potent inhibitor for SPases (6.0% relative activity at 4.5???M) but ferulic acid inhibits its activity at a much lower concentration (0.02???M). Enzymes were stimulated by metal cations like Cu²⁺, Ca²⁺ (145, 168; percentage relative activity respectively) and showed mild inhibition (up to 20%) with Mn²⁺ and Mg²⁺. Alanine stimulated the enzyme activity (up to 33%; at 0?C100???M) while other amino acids like cysteine, methionine, tryptophan and tyrosine inhibited the SPases (13?C57% at 0?C100???M).     

O2− from elicitor-induced oxidative burst is necessary for triggering phenylalanine ammonia-lyase activation and catharanthine synthesis in Catharanthus roseus cell cultures

Elicitor, derived from the cell walls of Aspergillus niger, induced rapid generation of reactive oxygen intermediates (ROI), including superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), sequentially followed by phenylalanine ammonia-lyase (PAL) activation and catharanthine biosynthesis in Catharanthus roseus suspension cells. The elicitor-induced PAL activation and catharanthine biosynthesis were blocked by NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI). O₂⁻ generated by the reaction of xanthine/xanthine oxidase (X/XO) triggered PAL activation and catharanthine biosynthesis of C. roseus cells in the absence of elicitor and reversed the inhibitory effect of DPI on elicitor-induced PAL activation and catharanthine biosynthesis. External application of H₂O₂ and catalase had no effect on PAL activity and catharanthine contents of C. roseus cells. The results demonstrated a causal relationship between elicitor-induced oxidative burst and PAL activation in C. roseus suspension cells and suggested a sequence of signaling events from ROI production to PAL activation and catharanthine synthesis. Within this sequence, O₂⁻ rather than H₂O₂ appeared to trigger the subsequent reactions.     

Purification and characterization of a catalase from the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 and its role in the oxidative stress response

When challenged with reactive oxidants, the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 exhibited an oxidative stress response during both phototrophic and chemotrophic growth. Upon preincubation with 100 μM H₂O₂, catalase activity increased fivefold. Catalase was also induced by other forms of oxidative stress, heat-shock, ethanol treatment, and stationary-phase conditions. Only one band of catalase activity was detected after native and denaturing PAGE. The enzyme was purified 304-fold with a yield of 7%. The purified enzyme displayed a heterodimeric structure with subunits of 75 and 68 kDa, corresponding to a molecular mass of approximately 150 kDa for the native enzyme. The subunits had almost identical amino-terminal peptide sequences, sharing substantial similarity with other bacterial catalases. The enzyme exhibited an apparent K _m of 40 mM and a V _max of 285,000 U (mg protein)^–1. Spectroscopic analysis indicated the presence of protoheme IX. The heme content calculated from pyridine hemochrome spectra was 0.43 mol per mol of enzyme. The enzyme had a broad pH optimum and was inhibited by cyanide, azide, hydroxylamine, 2-mercaptoethanol, and sodium dithionite. These data indicate that this catalase belongs to the class of monofunctional catalases. Received: 15 October 1997 / Accepted: 2 February 1998     

Manganoporphyrins and ascorbate enhance gemcitabine cytotoxicity in pancreatic cancer

Pharmacological ascorbate (AscH⁻) selectively induces cytotoxicity in pancreatic cancer cells vs normal cells via the generation of extracellular hydrogen peroxide (H₂O₂), producing double-stranded DNA breaks and ultimately cell death. Catalytic manganoporphyrins (MnPs) can enhance ascorbate-induced cytotoxicity by increasing the rate of AscH⁻ oxidation and therefore the rate of generation of H₂O₂. We hypothesized that combining MnPs and AscH⁻ with the chemotherapeutic agent gemcitabine would further enhance pancreatic cancer cell cytotoxicity without increasing toxicity in normal pancreatic cells or other organs. Redox-active MnPs were combined with AscH⁻ and administered with or without gemcitabine to human pancreatic cancer cell lines, as well as immortalized normal pancreatic ductal epithelial cells. The MnPs MnT2EPyP (Mn(III)meso-tetrakis(N-ethylpyridinium-2-yl) porphyrin pentachloride) and MnT4MPyP (Mn(III)tetrakis(N-methylpyridinium-4-yl) porphyrin pentachloride) were investigated. Clonogenic survival was significantly decreased in all pancreatic cancer cell lines studied when treated with MnP + AscH⁻ + gemcitabine, whereas nontumorigenic cells were resistant. The concentration of ascorbate radical (Asc^•−, an indicator of oxidative flux) was significantly increased in treatment groups containing MnP and AscH⁻. Furthermore, MnP + AscH⁻ increased double-stranded DNA breaks in gemcitabine-treated cells. These results were abrogated by extracellular catalase, further supporting the role of the flux of H₂O₂. In vivo growth was inhibited and survival increased in mice treated with MnT2EPyP, AscH⁻, and gemcitabine without a concomitant increase in systemic oxidative stress. These data suggest a promising role for the use of MnPs in combination with pharmacologic AscH⁻ and chemotherapeutics in pancreatic cancer.     

Examination of the superoxide/hydrogen peroxide forming and quenching potential of mouse liver mitochondria

Pyruvate dehydrogenase (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) are important sources of reactive oxygen species (ROS). In addition, it has been found that mitochondria can also serve as sinks for cellular hydrogen peroxide (H₂O₂). However, the ROS forming and quenching capacity of liver mitochondria has never been thoroughly examined. Here, we show that mouse liver mitochondria use catalase, glutathione (GSH), and peroxiredoxin (PRX) systems to quench ROS. Incubation of mitochondria with catalase inhibitor 3-amino-1,2,4-triazole (triazole) induced a significant increase in pyruvate or α-ketoglutarate driven O₂⁻/H₂O₂ formation. 1-Choro-2,4-dinitrobenzene (CDNB), which depletes glutathione (GSH), elicited a similar effect. Auranofin (AF), a thioredoxin reductase-2 (TR2) inhibitor which disables the PRX system, did not significantly change O₂⁻/H₂O₂ formation. By contrast catalase, GSH, and PRX were all required to scavenging extramitochondrial H₂O₂. In this study, the ROS forming potential of PDHC, KGDHC, Complex I, and Complex III was also profiled. Titration of mitochondria with 3-methyl-2-oxovaleric acid (KMV), a specific inhibitor for O₂⁻/H₂O₂ production by KGDHC, induced a ~ 86% and ~ 84% decrease in ROS production during α-ketoglutarate and pyruvate oxidation. Titration of myxothiazol, a Complex III inhibitor, decreased O₂⁻/H₂O₂ formation by ~ 45%. Rotenone also lowered ROS production in mitochondria metabolizing pyruvate or α-ketoglutarate indicating that Complex I does not contribute to ROS production during forward electron transfer from NADH. Taken together, our results indicate that KGDHC and Complex III are high capacity sites for O₂⁻/H₂O₂ production in mouse liver mitochondria. We also confirm that catalase plays a role in quenching either exogenous or intramitochondrial H₂O₂.     

Macrophage-mediated cytolysis of erythrocytes in the guinea pig. II. Role of oxidative burst products in macrophage cytotoxicity activated by lectins and phorbol ester derivatives

Guinea pig peritoneal macrophages (GPPM) exhibited enhanced production of O₂^? and H₂O₂, and cytolytic activity toward erythrocytes, in response to reagents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), its methylated derivative 4-O-MeTPA, Con A, wheat germ agglutinin (WGA), and opsonized zymosan. In order to examine the possible role of oxidative burst products such as O₂^? and H₂O₂ in the cytolytic process, we used reagents and enzymes which influence the balance of O₂^? and H₂O₂ outside and inside the GPPM cells. Macrophage-mediated cytolysis (MMC) of erythrocytes in the presence of the activators and modulators was assessed by ⁵¹Cr release assay. MMC activated by TPA and 4-O-MeTPA was inhibited by scavengers of H₂O₂ such as catalase and α-tocopherol, and was augmented by the catalase inhibitor 3-amino-1,2,4-triazole, and by horseradish peroxidase. TPA- and 4-O-MeTPA-activated MMC was only partially inhibited by the O₂^? scavenger cytochrome c and the enzyme superoxide dismutase and unaffected by cytochalasin D (an inhibitor of phagocytosis). MMC activated by the lectins Con A and WGA was unaffected by the scavengers and enzymes used, but markedly inhibited by cytochalasin D. Activation of MMC by TPA, WGA, and phagocytosis of opsonized zymosan, as well as O₂^? and H₂O₂ generation triggered by these reagents, were markedly inhibited by chlorpromazine. The results indicate that GPPM-mediated cytolysis activated by lectins, phorbol ester derivatives, and phagocytosis of opsonized zymosan, is dependent on the generation of oxidative burst products, mainly H₂O₂. TPA- or 4-O-MeTPA-activated MMC is mainly an extracellular event, while lectin-activated MMC may take place within the macrophages.     

Pathways of ROS homeostasis regulation in Mesembryanthemum crystallinum L. calli exhibiting differences in rhizogenesis

A comparison of the hydrogen peroxide (H₂O₂) content, proline and betacyanin concentration and activities of some antioxidant enzymes (catalase, superoxide dismutase, guaiacol and ascorbate peroxidases) was made in Mesembryanthemum crystallinum L. calli differing in rhizogenic potential. Callus was induced from hypocotyls of 10-day-old seedlings on a medium containing 1?mg?l^?1 2,4-dichlorophenoxyacetic acid and 0.2?mg?l^?1 kinetin, which was either supplemented with 40?mM NaCl (CIM-NaCl medium) or did not contain any salt (CIM medium). The callus obtained on CIM-NaCl was rhizogenic, whereas the callus induced on the medium without salt was non-rhizogenic throughout the culture. The rhizogenic callus differed from the non-rhizogenic callus in lower betacyanin and H₂O₂ content, but the rhizogenic callus displayed a higher proline level. The activity of H₂O₂ scavenging enzymes, such as catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (POD), was markedly higher in the rhizogenic callus than in the non-rhizogenic callus, but the total activity of superoxide dismutase (SOD) was higher in the non-rhizogenic callus than in the rhizogenic callus. Aminotriazole (CAT inhibitor) and diethyldithiocarbamate (SOD inhibitor) were added solely to the CIM and CIM-NaCl media to manipulate the concentration of reactive oxygen species (ROS) in the cultured tissues. Both CAT and SOD inhibitors brought about an increase in H₂O₂ content in calli cultured on CIM-NaCl and the loss of rhizogenic potential. Conversely, the addition of inhibitors to the medium without salt led to a decrease in H₂O₂ content. This corresponded with a significant decrease in the endogenous concentration of betacyanins, but did not change the lack of rhizogenic ability.     

A novel hydrogen peroxide sensor based on immobilization of haemoglobin on nano-TiO2/DTAB composite film

Abstract

The direct electron transfer of immobilized haemoglobin (Hb) on nano-TiO₂ and dodecyltrimethylammonium bromide (DTAB) film modified carbon paste electrode (CPE) and its application as a hydrogen peroxide (H₂O₂) biosensor were investigated. On nano-TiO₂/DTAB/Hb/CPE, Hb displayed a rapid electron transfer process with participation of one proton and with an electron transfer rate constant which estimated as 0.29 s^??1. Thus, the proposed biosensor exhibited a high sensitivity and excellent electrocatalytic activity for the reduction of H₂O₂. The catalytic reduction current of H₂O₂ was proportional to H₂O₂ concentration in the range of 0.2–4.0 mM with a detection limit of 0.07 mM. The apparent Michaelis–Menten constant (K_m^app) of the biosensor was calculated to be 0.127 mM, exhibiting a high enzymatic activity and affinity. This sensor for H₂O₂ can potentially be applied in determination of other reactive oxygen species as well.     

Enzymatic production of α-dehydrobiotin from biotin

The enzymatic production of α-dehydrobiotin (α-DHB), an antibiotic, from biotinyl-CoA using acyl-CoA oxidase and from biotin using a coupling system of biotinyl-CoA synthetase and acyl-CoA oxidase was developed. Acyl-CoA oxidase was found to show activity for biotinyl-CoA. K_m and V_max values of acyl-CoA oxidase for biotinyl-CoA were 75 μM and 3.92 μmol min⁻¹ mg⁻¹, respectively. Optimum reaction conditions for the α-DHB production from biotin were examined. The maximum production of α-DHB (4.29 μmol ml⁻¹) was obtained, when the reaction was carried out at 30°C for 36 h in a mixture consisting of 100 mM potassium phosphate buffer (pH 8.0), 20 mM biotin, 20 mM ATP, 60 mM CoA, 20 mM MgCl₂, 2 units of biotinyl-CoA synthetase, 90 units of acyl-CoA oxidase and 25 units of catalase in a total volume of 0.6 ml under aerobic conditions. The product was purified from 14 ml of the reaction mixture and 10 mg of crystals with white needle form were obtained. From NMR, mass spectra and other physical analyses, this compound was identified as (+)-trans-α-DHB.     

Determination of catalase activity using chromogenic probe involving iso-nicotinicacidhydrazide and pyrocatechol

A biocatalatic pathway involving chromogenic probe has been proposed for the determination of catalase activity by means of iso-nicotinicacidhydrazide (INH) and pyrocatechol (PC). The assay is based on the enzymatic consumption of hydrogen peroxide using INH-PC system. The response of the catalase activity was ascertained by the rate of the reaction involving 14.10 mM H₂O₂. On addition of H₂O₂, INH-PC indicator system formed a chromogenic product with absorbance maxima at 490 nm. Hence the activity of catalase was directly measured by the chromogenic response in the formation of the coupled product. The catalase assay was elaborated by the kinetic response of the INH-PC system. The linearity of the catalase activity and H₂O₂ was in the range 0.2-7.0 units and 1.76-7.0 mM, respectively in 3 ml solution. The catalytic efficiency and catalytic power were calculated. The Michaelis-Menten constant of INH, PC and H₂O₂ were found to be 0.344, 0.176 and 8.82 mM, respectively. The indicator reaction was applied in the determination of catalase activity in mycelia mats and culture media.     

Role of hydrogen peroxide and apoplastic peroxidase in tomato — Botrytis cinerea interaction

The aim of the research was to estimate the sensitivity of tomato tissue and spore from necrotrophic isolate of B. cinerea on H₂O₂. The influence of exogenic H₂O₂ and B. cinerea on plant tissue and on the activity of peroxidases (PO), catalase (CAT) and superoxide dismutase (SOD) in apoplastic tomato leaves fraction were investigated. It was proved that 40 mM H₂O₂ damaged the cells of a host, and inhibited in vitro germination of B.cinerea spores. Complete inhibition of germination was observed after the use 100 mM H₂O₂. In the presence of spores H₂O₂ was decomposed to H₂O and O₂. Trace activity of catalase was observed in a solution of spores used for inoculation. Necrosis which appeared on the leaves after 40 mM H₂O₂ treatment resembled hypersensitive response. On the leaves pretreated at this concentration the development of infection was observed. The H₂O₂ concentration harmful for the tissues, stimulated the PO activity measured with NADH — responsible for generation of ^·O ₂ ⁻ , as well as with syringaldazine (S) and ferulic acid (FA), substrates characteristics of forms lignifying and strengthening the cell wall. Clear increase in CAT activity, resulting from infection and early pretreatment with H₂O₂ was observed in apoplast. No effect on SOD activity was observed. A hypothesis may be put forward, that germinating spores produce enzymes which allow them to decompose H₂O₂ generated in apoplast, so there is little likelihood that B. cinerea can be directly inhibited by reactive oxygen forms (ROS) during initial stages of infection. Necrotic lesions resembling HR generated by exogenous H₂O₂ as well as induction of activity of apoplastic plant enzymes, particularly PO connected with strengthening and lignification of cell wall, were not sufficient factors to inhibit fungal expansion.     

Nitrogen deprivation induces cross-tolerance of Poa annua callus to salt stress

Alternative respiration pathway (AP) is an important pathway which can be induced by environment stresses in plants. In the present study, we show a new mechanism involving the AP in nitrogen deprivation-induced tolerance of Poa annua callus to salt stress. The AP capacity markedly increased under a 600 mM NaCl treatment or nitrogen deprivation pretreatment and reached a maximum under the nitrogen deprivation pretreatment combined with the NaCl treatment (–N+NaCl). Malondialdehyde (MDA) and H₂O₂ content and Na⁺/K⁺ ratio significantly increased under the 600 mM NaCl treatment but less under the–N+NaCl treatment. Moreover, both the nitrogen deprivation and the NaCl stress stimulated the plasma membrane (PM) H⁺-ATPase activity and increased pyruvate content. The maximal stimulating effect was found under the–N+NaCl treatment. When the AP capacity was reduced by salicylhydroxamic acid (SHAM, an inhibitor of AP), content of MDA and H₂O₂ and Na⁺/K⁺ ratio dramatically increased, whereas PM H⁺-ATPase activity decreased. Moreover, exogenous application of pyruvate produced a similar effect as the nitrogen deprivation pretreatment. The effects of SHAM on the Poa annua callus were counteracted by catalase (a H₂O₂ scavenger) and diphenylene iodonium (a plasma membrane NADPH oxidase inhibitor). Taken together, our results suggest that the nitrogen deprivation enhanced the capacity of AP by increasing pyruvate content, which in turn prevented the Poa annua callus from salt-induced oxidative damages and Na⁺ over-uptake.