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1.
Empirical estimations of H 2O 2 concentration in a system containing bovine liver catalase and continually supplied with H 2O 2 were done to evaluate the efficiency of the enzyme to cleave H 2O 2. It was found that the continuous addition of H 2O 2 leads to the formation of steady-state concentrations of H 2O 2 in the medium. At a constant catalase concentration both the level and the duration of the steady state are dependent on the flow rate of H 2O 2. The increase of the catalase concentration in the medium does not change the steady-state level, it merely leads to the maintenance of the steady state for longer durations. At higher flow rates of H 2O 2, no steady state could be maintained, even when catalase was present in high excess. The incomplete cleavage of H 2O 2 by catalase under these conditions is due to the low affinity of catalase toward H 2O 2 (high Km value, apparent Km = 0.1 M H 2O 2) and to the rapid inactivation of the enzyme during the continuous addition of H 2O 2. 相似文献
2.
The nonenzymatic reaction of glyoxylate and H 2O 2 was measured under physiological conditions of the pH and concentrations of reactants. The reaction of glyoxylate and H 2O 2 was secondorder, with a rate constant of 2.27 l mol -1 s -1 at pH 8.0 and 25° C. The rate constant increased by 4.4 times in the presence of Zn 2+ and doubled at 35°C. We propose a mechanism for the reaction between glyoxylate and H 2O 2. From a comparison of the rates of H 2O 2 decomposition by catalase and the reaction with glyoxylate, we conclude that H 2O 2 produced during glycolate oxidation in peroxisomes is decomposed by catalase but not by the reaction with glyoxylate, and that photorespiratory CO 2 originates from glycine, but not from glyoxylate, in C 3 plants. Simulation using the above rate constant and reported kinetic parameters leads to the same conclusion, and also makes it clear that alanine is a satisfactory amino donor in the conversion of glyoxylate to glycine. Some serine might be decomposed to give glycine and methylene-tetrahydrofolate; the latter is ultimately oxidized to CO 2. In the simulation of the glycolate pathway of Euglena, the rate constant was high enough to ensure the decarboxylation of glyoxylate by H 2O 2 to produce photorespiratory CO 2 during the glycolate metabolism of this organism.Abbreviations Chl
chlorophyll
- GGT
glutamate: glyoxylate aminotransferase (EC 2.6.1.4)
- Hepes
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- SGT
serine: glyoxylate aminotransferase (EC 2.6.1.45)
This is the ninth in a series on the metabolism of glycolate in Euglena gracilis. The eighth is Yokota et al. (1982) 相似文献
3.
The increase in net photosynthesis in M 4 progeny of an O 2-resistant tobacco ( Nicotiana tabacum) mutant relative to wild-type plants at 21 and 42% O 2 has been confirmed and further investigated. Self-pollination of an M 3 mutant produced M 4 progeny segregating high catalase phenotypes (average 40% greater than wild type) at a frequency of about 60%. The high catalase phenotype cosegregated precisely with O 2-resistant photosynthesis. About 25% of the F 1 progeny of reciprocal crosses between the same M 3 mutant and wild type had high catalase activity, whether the mutant was used as the maternal or paternal parent, indicating nuclear inheritance. In high-catalase mutants the activity of NADH-hydroxypyruvate reductase, another peroxisomal enzyme, was the same as wild type. The mutants released 15% less photorespiratory CO 2 as a percent of net photosynthesis in CO 2-free 21% O 2 and 36% less in CO 2-free 42% O 2 compared with wild type. The mutant leaf tissue also released less 14CO 2 per [1- 14C]glycolate metabolized than wild type in normal air, consistent with less photorespiration in the mutant. The O 2-resistant photosynthesis appears to be caused by a decrease in photorespiration especially under conditions of high O 2 where the stoichiometry of CO 2 release per glycolate metabolized is expected to be enhanced. The higher catalase activity in the mutant may decrease the nonenzymatic peroxidation of keto-acids such as hydroxypyruvate and glyoxylate by photorespiratory H 2O 2. 相似文献
4.
Summary Intact chloroplasts capable of high rates of CO 2 assimilation completely oxidized 3-phosphoglycerate and dihydroxyacetone phosphate to glycolate when CO 2 concentrations were low. Bicarbonate was converted first into products of the Calvin cycle and then into glycolate. Under high oxygen and at high pH values CO 2 fixation and glycolate formation ceased before bicarbonate was exhausted. This is interpreted as the consequence of a depletion of ribulose diphosphate (RuDP) at the oxygen compensation point, where oxygen consumption by glycolate formation and oxygen evolution by phosphoglycerate reduction balance each other. Depletion of RuDP by glycolate formation is proposed to play a role in the Warburg effect. The maximum rate of glycolate synthesis observed with dihydroxyacetone phosphate as substrate was 35 mol mg -1 chlorophyll h -1 at 20°C. This may not reflect the maximum capacity of chloroplasts for glycolate synthesis. Dithiothreitol and catalase, which prevent accumulation of oxygen radicals or H 2O 2 during carbon assimilation, increased glycolate formation. H 2O 2 was inhibitory. Other inhibitors of glycolate formation were glyceraldehyde and carbonylcyanide p-trifluoro-methoxphenylhydrazone. From the sensitivity of glycolate synthesis to uncoupling and the ATP requirement of RuDP formation it is concluded that glycolate originated from RuDP. Different induction periods of carbon fixation and glycolyte formation suggested that glycolate synthesis is not only regulated by the ratio of oxygen to CO 2 but also by another factor. 相似文献
5.
A reconstituted spinach chloroplast system containing thylakoids, stroma and 0.1 mM NADPH supported O 2 evolution in the presence of oxidised glutathione (GSSG). The properties of the reaction were consistent with light-coupled GSSG-reductase activity involving H 2O as eventual electron donor. The reconstituted system also supported dehydroascorbate-dependent O 2 evolution in the presence of 0.6 mM reduced glutathione (GSH) and 0.1 mM NADPH with the concomitant production of ascorbate. The GSSG could replace GSH in which case the production of GSH preceded the accumulation of ascorbate. The data are consistent with the light-dependent reduction of dehydroascorbate using H 2O as eventual electron donor via the sequence H 2O→NADP→GSSG→dehydroascorbate. Approximately 30% of the GSH-dehydrogenase activity of spinach leaf protoplasts is localised in chloroplasts: this could not be attributed to contamination of chloroplasts by activity from the extrachloroplast compartment. Washed intact chloroplasts supported the uptake of ascorbate but the uptake mechanism had a very low affinity for ascorbate (K m approximately 20 mM). The rate of uptake of ascorbate was less than the rate of light-dependent reduction of dehydroascorbate and too slow to account for the rate of H 2O 2 reduction by washed intact chloroplasts. 相似文献
6.
Decreasing substrate osmotic potential produced in seedlings of Vigna catjang Endl. (cv. Pusa Barsati) proportional decrease in relative water content and leaf water potential, increase in respiration rate, proline content, H 2O 2 content, and the activities of indole acetic acid oxidase, ascorbic acid oxidase, peroxidase and glycolate oxidase but decrease in catalase activity and glycolate content. Pretreatment with reducing agents like L-cysteine or reduced glutathione (10?3 M) caused lower decrease in the relative water content, leaf water potential and glycolate content and reduced the rise of respiration rate, proline content and H 2O 2 content and also the activities of aforementioned oxidative enzymes, except catalase activity which was increased. Such treatments also maintained the chlorophyll and protein levels and decreased the tissue permeability. It was concluded that the treatment of Vigna seedlings with reducing agents reduced the deteriorative changes and oxidative processes which are characteristic of water stressed tissue. 相似文献
7.
The physiological role of peroxisomal ascorbate peroxidases (pAPX) is unknown; therefore, we utilized pAPX4 knockdown rice and catalase (CAT) inhibition to assess its role in CAT compensation under high photorespiration. pAPX4 knockdown induced co‐suppression in the expression of pAPX3. The rice mutants exhibited metabolic changes such as lower CAT and glycolate oxidase (GO) activities and reduced glyoxylate content; however, APX activity was not altered. CAT inhibition triggered different changes in the expression of CAT, APX and glutathione peroxidase (GPX) isoforms between non‐transformed (NT) and silenced plants. These responses were associated with alterations in APX, GPX and GO activities, suggesting redox homeostasis differences. The glutathione oxidation‐reduction states were modulated differently in mutants, and the ascorbate redox state was greatly affected in both genotypes. The pAPX suffered less oxidative stress and photosystem II (PSII) damage and displayed higher photosynthesis than the NT plants. The improved acclimation exhibited by the pAPX plants was indicated by lower H 2O 2 accumulation, which was associated with lower GO activity and glyoxylate content. The suppression of both pAPXs and/or its downstream metabolic and molecular effects may trigger favourable antioxidant and compensatory mechanisms to cope with CAT deficiency. This physiological acclimation may involve signalling by peroxisomal H 2O 2, which minimized the photorespiration. 相似文献
8.
Extended exposure of Escherichia coli to temperatures above and below their growth optimum led to significant changes in oxidant production and antioxidant defense. At 20 °C an increase in the intracellular H 2O 2 concentration and oxidized glutathione (GSSG) level was observed against a background of low levels of reduced glutathione (GSH) and decreased catalase and glutathione reductase (GOR) activities. The intracellular H 2O 2 and GSSG concentrations had minimal values at 30 and 37 °C, but rose again at 42 °C, suggesting that oxidative processes were intensified at high temperatures. An increase in temperature from 20 to 42 °C led to an elevation in the oxygen respiration rate and superoxide production; a 5-fold increase in the intracellular GSH concentration and in the GSH:GSSG ratio occurred simultaneously. Catalase HPI and GOR activities were elevated 4.4- and 1.5-fold, respectively. Prolonged exposure to sublethal temperatures facilitated an adaptation to subsequent oxidative stress produced by the addition of H 2O 2. 相似文献
9.
Although active oxygen species are produced at high rates inboth the chloroplasts and peroxisomes of the leaves of C 3 plants,most attention has focused on the potentially damaging consequencesof enhanced chloroplastic production in stress conditions suchas drought. This article attempts to provide quantitative estimatesof the relative contributions of the chloroplast electron transportchain and the glycolate oxidase reaction to the oxidative loadplaced on the photosynthetic leaf cell. Rates of photorespiratoryH 2O 2 production were obtained from photosynthetic and photorespiratoryflux rates, derived from steady-state leaf gas exchange measurementsat varying irradiance and ambient CO 2. Assuming a 10 % allocationof photosynthetic electron flow to the Mehler reaction, photorespiratoryH 2O 2 production would account for about 70 % of total H 2O 2 formedat all irradiances measured. When chloroplastic CO 2 concentrationrates are decreased, photorespiration becomes even more predominantin H 2O 2 generation. At the increased flux through photorespirationobserved at lower ambient CO 2, the Mehler reaction would haveto account for more than 35 % of the total photosynthetic electronflow in order to match the rate of peroxisomal H 2O 2 production.The potential signalling role of H 2O 2 produced in the peroxisomesis emphasized, and it is demonstrated that photorespiratoryH 2O 2 can perturb the redox states of leaf antioxidant pools.We discuss the interactions between oxidants, antioxidants andredox changes leading to modified gene expression, particularlyin relation to drought, and call attention to the potentialsignificance of photorespiratory H 2O 2 in signalling and acclimation. 相似文献
10.
Mycoplasma iowae is a well-established avian pathogen that can infect and damage many sites throughout the body. One potential mediator of cellular damage by mycoplasmas is the production of H 2O 2 via a glycerol catabolic pathway whose genes are widespread amongst many mycoplasma species. Previous sequencing of M. iowae serovar I strain 695 revealed the presence of not only genes for H 2O 2 production through glycerol catabolism but also the first documented mycoplasma gene for catalase, which degrades H 2O 2. To test the activity of M. iowae catalase in degrading H 2O 2, we studied catalase activity and H 2O 2 accumulation by both M. iowae serovar K strain DK-CPA, whose genome we sequenced, and strains of the H 2O 2-producing species Mycoplasma gallisepticum engineered to produce M. iowae catalase by transformation with the M. iowae putative catalase gene, katE. H 2O 2-mediated virulence by M. iowae serovar K and catalase-producing M. gallisepticum transformants were also analyzed using a Caenorhabditis elegans toxicity assay, which has never previously been used in conjunction with mycoplasmas. We found that M. iowae katE encodes an active catalase that, when expressed in M. gallisepticum, reduces both the amount of H 2O 2 produced and the amount of damage to C. elegans in the presence of glycerol. Therefore, the correlation between the presence of glycerol catabolism genes and the use of H 2O 2 as a virulence factor by mycoplasmas might not be absolute. 相似文献
11.
Rat liver peroxisomes are membrane-bounded organelles containing catalase and oxidases producing H 2O 2. Diffusion effects in the metabolism of H 2O 2 and the physiological significance of the structure of peroxisomes are explored on the basis of two models. Model I considers the liver cell as consisting of two rapidly mixed compartments, the peroxisomal contents and the rest of the cell, separated by a membrane. On the basis of model I, it is concluded that in order to maintain a minimal H 2O 2 concentration in the cytoplasm, there must be an H 2O 2 destroying system in the cytoplasm, but the capacity of this system need be only a small fraction of that of the catalase in the peroxisomes. Model II takes account of the detailed morphology of peroxisomes and includes the effect of peroxisomal membrane permeability to H 2O 2 and H 2O 2 diffusion inside and outside the peroxisomes. On the basis of previously published experimental data and model II, it is concluded that the latency of catalase activity in intact peroxisomes is due chiefly to a permeability barrier to H 2O 2 at the peroxisomal membrane rather than to a restriction of H 2O 2 diffusion within the peroxisomes. Peroxisomes are calculated to be very efficient at destroying the H 2O 2 produced within them, whether the H 2O 2 is produced in the catalase-free core or in the catalase-containing matrix. Less than 2% of the H 2O 2 produced in peroxisomes leaves the particles. The efficiency of H 2O 2 trapping is the consequence of the membrane permeability barrier. A similar H 2O 2 trapping efficiency could be achieved by particles without a membrane barrier only if H 2O 2 diffusion within such particles were reduced by many orders of magnitude. 相似文献
13.
In order to understand the continuous defense reactions of host plants against insect attack, a tobacco variety G140 was infested by tobacco aphid Myzus persicae for 2 h to 5 d. The changes of transmembrane ionic fluxes (Ca 2+) and hydrogen peroxide (H 2O 2) were detected by the technique of noninvasive micro-test and their relationship was further studied. It was found that H 2O 2 accumulation depended on Ca 2+ influx. Ca 2+ flux exhibited a strong influx at all infestation periods by aphids, while H 2O 2 showed an efflux behavior. The slight variation tendency of Ca 2+ influx and H 2O 2 efflux was consistent. The activities of the corresponding defense proteins, peroxidase (POD) and catalase (CAT) enzyme, were enhanced to respond to the insect attacks, much higher than those tobacco in control. The Ca 2+ influx and H 2O 2 efflux, as well as the activities of POD and CAT enzymes, were increased in a long period of aphid feeding. It indicated that a continuous physiological response of tobacco to aphid infestation could be initiated and lasted for a long time. 相似文献
14.
Intracellular catalase activity was measured in isolated rat hepatocytes by adding H 2O 2 under anaerobic conditions and measuring O 2 evolution. Hydrogen peroxide was introduced either by continuous infusion or by pulse injection. Continuous infusion at a rate similar to the endogenous H 2O 2 production rate provided results that 60–70% of the H 2O 2 was metabolized by the catalatic reaction. Comparison of rates of O 2 evolution to estimated rates of H 2O 2 metabolism obtained by the methanol-titration method ( H. Sies and B. Chance, 1970, FEBS Lett.11, 172–176) indicated that the contribution of the peroxidatic reaction of catalase was small. The intracellular activity of glutathione peroxidase was estimated as the catalase-independent metabolism and used to determine the rate of intracellular H 2O 2 metabolism by the peroxidase. The results provide a quantitative basis for analysis of the physiological and toxicological aspects of H 2O 2 metabolism by liver. 相似文献
15.
Oxidative stress is a major challenge for all cells living in an oxygen‐based world. Among reactive oxygen species, H 2O 2, is a well known toxic molecule and, nowadays, considered a specific component of several signalling pathways. In order to gain insight into the roles played by H 2O 2 in plant cells, it is necessary to have a reliable, specific and non‐invasive methodology for its in vivo detection. Hence, the genetically encoded H 2O 2 sensor HyPer was expressed in plant cells in different subcellular compartments such as cytoplasm and peroxisomes. Moreover, with the use of the new green fluorescent protein (GFP)‐based Cameleon Ca 2+ indicator, D3cpv–KVK–SKL, targeted to peroxisomes, we demonstrated that the induction of cytoplasmic Ca 2+ increase is followed by Ca 2+ rise in the peroxisomal lumen. The analyses of HyPer fluorescence ratios were performed in leaf peroxisomes of tobacco and pre‐ and post‐bolting Arabidopsis plants. These analyses allowed us to demonstrate that an intraperoxisomal Ca 2+ rise in vivo stimulates catalase activity, increasing peroxisomal H 2O 2 scavenging efficiency. 相似文献
16.
When leaf discs of Xanthium strumarium L. and Salvia splendens L. are incubated in sealed flasks in the light, more C 2H 4 gas is released in the presence of added CO 2 (30-200 millimolar NaHCO 3) than without CO 2. In Salvia, the maximum rate of C 2H 4 release occurs when sufficient CO 2 (above 125 millimolar NaHCO 3) is added to saturate photosynthesis confirming previous studies. The maximum rate of C 2H 4 release from illuminated discs is similar to the rate in the dark with or without CO 2 in both species. Glycolate enhances a CO 2-dependent C 2H 4 evolution from illuminated leaf discs. However, the maximum rate of C 2H 4 release with glycolate is the same as that observed with saturating CO 2. When photosynthesis is inhibited by darkness or by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, glycolate has no effect. Studies with [2,3-14C]-1-aminocyclopropane-1-carboxylic acid (ACC) show that the pattern of C2H4 release and the specific activity of the 14C2H4 in the presence and absence of glycolate is similar to that described above, indicating that glycolate does not alter uptake of the exogenously supplied precursor (ACC) or stimulate C2H4 release from an endogenous source at appreciable rates. Glycolate oxidase in vitro generates H2O2 which stimulates a slow breakdown of ACC to C2H4, but since exogenous glycolate is oxidized to CO2 in both the light and the dark it is argued that the glycolate-dependent increase in C2H4 release from illuminated leaf discs is not mediated directly by the action of enzymes of glycolate catabolism. The effects of glycolate and CO2 are not easily explained by changes in stomatal resistance. The data support the view that glycolate decarboxylation at subsaturating levels of CO2 in the light stimulates C2H4 release by raising the CO2 level in the tissue. 相似文献
17.
In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H 2O 2) through the plasma membrane decreases during adaptation to H 2O 2 by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H 2O 2 the anisotropy of the plasma membrane increases. Adaptation to H 2O 2 was studied at several times (15min up to 90min) by applying the steady-state H 2O 2 delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H 2O 2 was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H 2O 2 adaptation. H 2O 2 diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H 2O 2 permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H 2O 2 is mediated by modulation of the biophysical properties of the plasma membrane. 相似文献
18.
As a boy, I read Sinclair Lewis's Arrowsmithand dreamed of doing research of potential benefit to society. I describe the paths of my scientific career that followed.
Several distinguished scientists served as my mentors and I present their profiles. Much of my career was in a small department
at a small institution where independent researchers collaborated informally. I describe the unique method of carrying on
research there. My curiosity about glycolate metabolism led to unraveling the enzymatic mechanism of the glycolate oxidase
reaction and showing the importance of H 2O 2 as a byproduct. I discovered enzymes catalyzing the reduction of glyoxylate and hydroxypyruvate. I found α-hydroxysulfonates
were useful competitive inhibitors of glycolate oxidase. In a moment of revelation, I realized that glycolate metabolism was
an essential part of photorespiration, a process that lowers net photosynthesis in C 3 plants. I added inhibitors of glycolate oxidase to leaves and showed: (1) glycolate was synthesized only in light as an early
product of photosynthetic CO 2 assimilation, (2) the rate of glycolate oxidation consumed a sizable fraction of net photosynthesis in C 3 but not in C 4 plants, and (3) that glycolate metabolism increased greatly at higher temperatures. For a while I studied the control of
stomatal opening in leaves, and this led to the finding that potassium ions are a key solute in guard cells. I describe experiments
that show that when photorespiration rates are high, as occurs at higher temperatures, genetically increasing leaf catalase
activity reduces photorespiration and increases net photosythetic CO 2 assimilation.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
19.
Conclusions and summary 1. Oxygen has two polarographic waves of equal height (h.w.p. –0.05 V and –1.07 V), which disturb the direct polarographic determination of H 2O 2.2. In determining H 2O 2 it is possible to eliminate this disturbance by reducing the total height of the waves by 2 × the height of the first O 2 wave.3. In a decomposing H 2O 2 solution the O 2 concentration exceeds more than 5 × the normal O 2 concentration in an aqueous solution.4. In a decomposing H 2O 2 solution the H 2O 2 concentration at each moment can only be determined by taking into account the O 2 concentration at that moment.5. Determination without this correction presents too small a catalase activity and may even result in characterising catalase positive bacteria as catalase negative.6. It is possible to demonstrate the H 2O 2 production of catalase negative bacteria by the polarographic method. 相似文献
20.
When glycolate was metabolized in peroxisomes isolated from leaves of spinach beet ( Beta vulgaris L., var. vulgaris) formate was produced. Although the reaction mixture contained glutamate to facilitate conversion of glycolate to glycine, the rate at which H 2O 2 became “available” during the oxidation of [1- 14C]glycolate was sufficient to account for the breakdown of the intermediate [1- 14C]glyoxylate to formate (C 1 unit) and 14CO 2. Under aerobic conditions formate production closely paralleled 14CO 2 release from [1- 14C]glycolate which was optimal between pH 8.0 and pH 9.0 and was increased 3-fold when the temperature was raised from 25 to 35 C, or when the rate of H 2O 2 production was increased artificially by addition of an active preparation of fungal glucose oxidase. 相似文献
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