Hymenolepis diminuta: Biochemical properties of peroxidase activity in mitochondria

The biochemical properties of a peroxidase previously localized cytochemically in the mitochondria of Hymenolepis diminuta were determined. The method chosen was the o-dianisidine procedure in which the decomposition of hydrogen peroxide has been followed spectrophotometrically. Peroxidase activity was initially demonstrated in the mitochondrial pellet. Subsequently, mitochondrial pellets were sonicated and the membrane and supernatant fractions were tested for peroxidase activity. Enzyme activity was demonstrated in the membrane fraction. The enzyme displayed a pH optimum of 5.0, was ascorbate sensitive, and was inhibited by excess H₂O₂. Neither peroxidase nor catalase were observed in any other fraction of the tapeworm tissue, confirming previous cytochemical investigations.     

Pseudomonas cytochrome c peroxidase Initial delay of the peroxidatic reaction. Electron transfer properties

A delay of some seconds is observed in the reaction of Pseudomonas cytochrome c peroxidase if the reaction is initiated by adding the enzyme to the reaction mixture containing reduced electron donor and hydrogen peroxide. This lag phase is avoided if the enzyme is incubated with the reduced electron donor and the reaction is started by adding hydrogen peroxide. The nature of the inital delay has been studied and it is shown that the peroxidase is reduced before a steady-state rate in the peroxidatic reaction is reached. The ability of the peroxidase to accept electrons from various electron donors emphasizes its cytochrome-like properties.     

Oxidative stress tolerance in the filamentous green algae Cladophora glomerata and Enteromorpha ahlneriana

Cladophora glomerata (L.) Kütz. and Enteromorpha ahlneriana Bliding are morphologically similar filamentous green algae that are dominants in the upper littoral zone of the brackish Baltic Sea. As these two species co-exist in a continuously fluctuating environment, we hypothesised that they may have different strategies to cope with oxidative stress. This was tested in laboratory experiments through stressing the algae by high irradiance (600 μmol photons PAR m⁻² s⁻¹) at two different temperatures (15 and 26 °C) in a closed system. Thus, oxidative stress was created by high irradiance (photo-oxidative stress) and/or carbon depletion. The extent of lipid oxidative damage, antioxidant enzyme activities and the amount of hydrogen peroxide excreted by the algae to the surrounding seawater medium were measured. The results suggest that the two species have different strategies: the annual C. glomerata could be classified as a more stress-tolerant species and the ephemeral E. ahlneriana as a more stress-susceptible species. Low temperature in combination with high irradiance created less lipid oxidative damage in C. glomerata than in E. ahlneriana, which was probably related to the higher regular activities of the hydrogen peroxide scavenging enzymes catalase and ascorbate peroxidase in C. glomerata, whereas in E. ahlneriana high activities of these enzymes were only obtained after the induction of oxidative stress. Superoxide dismutase activities were similar in both species, but the mechanisms to remove the hydrogen peroxide produced by the action of this enzyme were different: more through scavenging enzymes in C. glomerata and more through excretion to the seawater medium in E. ahlneriana. The high excretion of hydrogen peroxide, possibly in combination with brominated volatile halocarbons, by E. ahlneriana may have a negative effect on epiphytes and may partly explain why this alga is usually remarkably devoid of epiphytes and grazers compared to C. glomerata.     

The Escherichia coli btuE gene, encodes a glutathione peroxidase that is induced under oxidative stress conditions

Most aerobic organisms are exposed to oxidative stress. Looking for enzyme activities involved in the bacterial response to this kind of stress, we focused on the btuE-encoded Escherichia coli BtuE, an enzyme that shares homology with the glutathione peroxidase (GPX) family. This work deals with the purification and characterization of the btuE gene product.Purified BtuE decomposes in vitro hydrogen peroxide in a glutathione-dependent manner. BtuE also utilizes preferentially thioredoxin A to decompose hydrogen peroxide as well as cumene-, tert-butyl-, and linoleic acid hydroperoxides, confirming that its active site confers non-specific peroxidase activity. These data suggest that the enzyme may have one or more organic hydroperoxide as its physiological substrate.The btuE gene was induced when cells were exposed to oxidative stress elicitors that included potassium tellurite, menadione and hydrogen peroxide, among others, suggesting that BtuE could participate in the E. coli response to reactive oxygen species. To our knowledge, this is the first report describing a glutathione peroxidase in E. coli.     

The tyrosinase activity of melanosomes from the harding-passey melanoma: The absence of a peroxidase component in vitro

Melanosomes were isolated from the Harding-Passey melanoma with a density gradient technique. Using the Pomerantz radioassay for tyrosinase activity it was found that these isolated melanosomes could hydroxylate tyrosine in the presence of catalase sufficient to deny the enzyme any hydrogen peroxide. It was further found that the rate of hydroxylation was unaffected by the presence of exogenous hydrogen peroxide. Tyrosinase activity could be suppressed by preincubation in diethyldithiocarbamate followed by removal of this inhibitor before enzyme assay. Attempts to regain enzymatic activity, however, by addition of copper II ions were unsuccessful. No peroxidase activity could be detected on the isolated granules, and indeed evidence for a peroxidase inhibitor on the granules was found. It was also found that the peroxidase activity present in a 20% homogenate of mouse muscle did not demonstrate any tyrosinase activity with the Pomerantz assay even in the presence of hydrogen peroxide. It is concluded from these studies that there is tyrosinase on these melanosomes which is capable in vitro of hydroxylating tyrosine without any contribution from an active peroxidase.     

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus

Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O₂ and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O₂ exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.     

Induction of catalase in Escherichia coli by ascorbic acid involves hydrogen peroxide

Ascorbic acid at concentrations between 0.57 and 5.7 mM in aerated medium caused an eight fold increase in catalase activity in Escherchia coli. The hydrogen peroxide concentrations resulting from ascorbate oxidation were between 20 and 120 μM and hydrogen peroxide by itself caused a similar increase in catalase levels in both aerobic and anaerobic media. Three catalase activity bands visualized on polyacrylamide gels were increased. Chloramphenicol which inhibits protein synthesis, anaerobic medium and EDTA, which prevent ascorbate oxidation, and exogenous catalase, which removes hydrogen peroxide from the medium, all prevented the increase in catalase in response to ascorbate. Superoxide dismutase activity was not affected by ascorbate.     

Molecular cloning of Daphnia magna catalase and its biomarker potential against oxidative stresses

Catalase (EC 1.11.1.6) is an important antioxidant enzyme that protects aerobic organisms against oxidative damage by degrading hydrogen peroxide to oxygen and water. Catalase mRNAs have been cloned from many species and employed as useful biomarkers of oxidative stress. In the present study, we cloned the cDNA from the catalase gene in Daphnia magna, analyzed its catalytic properties, and investigated mRNA expression patterns after the exposure to known oxidative stressors. The catalase proximal heme-ligand signature sequence, FDRERISERVVHAKGSGA, and the proximal active site signature, RLFSYTDTH, are highly conserved. The variation of catalase mRNA expression in D. magna was quantified by real-time PCR, and the results indicated that catalase expression was up-regulated after exposure to UV-B light or cadmium (Cd). The activity of catalase enzyme also showed a similar increasing pattern when exposed to these model stressors. The full-length catalase cDNA of D. magna was cloned using mixed primers by the method of 3′ and 5′ rapid amplification of cDNA ends PCR. The cDNA sequence consists of 1515 nucleotides, encoding 504 amino acids. Sequence comparison showed that the deduced amino acid sequence of D. magna shared 73%, 72%, 71% and 70% identity with that of Chlamys farreri, Fenneropenaeus chinensis, Litopenaeus vannamei and Anopheles gambiae, respectively. This study shows that the catalase mRNA from D. magna could be successfully employed as a biomarker of oxidative stress, which is a common mode of toxicity for many water contaminants.     

Peroxidase activity in the brown alga Laminaria digitata

Cell-free extracts of the brown alga Laminaria digitata catalyse the oxidation of o-dianisidine and of iodide, as well as the formation of iodoamino acids. The enzyme(s) requires hydrogen peroxide for these activities, which are strongly inhibited by cyanide and azide. It is suggested that the activity may be due to a haem-containing peroxidase which, in extracts, is strongly bound, possibly to alginate.     

Regulation of glutamine synthetase by metal-catalyzed oxidative modification in the marine oxyphotobacterium Prochlorococcus

The inactivation of glutamine synthetase (GS; EC 6.3.1.2) by metal-catalyzed oxidation (MCO) systems was studied in several Prochlorococcus strains, including the axenic PCC 9511. GS was inactivated in the presence of various oxidative systems, either enzymatic (as NAD(P)H+NAD(P)H-oxidase+Fe³⁺+O₂) or non-enzymatic (as ascorbate+Fe³⁺+O₂). This process required the presence of oxygen and a metal cation, and is prevented under anaerobic conditions. Catalase and peroxidase, but not superoxide dismutase, effectively protected the enzyme against inactivation, suggesting that hydrogen peroxide mediates this mechanism, although it is not directly responsible for the reaction. Addition of azide (an inhibitor of both catalase and peroxidase) to the MCO systems enhanced the inactivation. Different thiols induced the inactivation of the enzyme, even in the absence of added metals. However, this inactivation could not be reverted by addition of strong oxidants, as hydrogen peroxide or oxidized glutathione. After studying the effect of addition of the physiological substrates and products of GS on the inactivation mechanism, we could detect a protective effect in the case of inorganic phosphate and glutamine. Immunochemical determinations showed that the concentration of GS protein significantly decreased by effect of the MCO systems, indicating that inactivation precedes the degradation of the enzyme.     

Experimental studies on the cadmium accumulation in the cestode Moniezia expansa (Cestoda: Anoplocephalidae) and its final host (Ovis aries)

The tapeworm Moniezia expansa and naturally infected sheep were investigated with respect to their cadmium accumulation. Cadmium chloride (CdCl₂, 0.2 g) was added to 10 ml of distilled water and administered orally to the sheep every day for a period of 1 week. The cadmium content of M. expansa was lower than that in the liver tissues of sheep, although this difference was not significant. The highest mean cadmium concentrations were found in the liver of sheep infected with M. expansa (24.5 ± 11.5 mg kg⁻¹ dry weight). The mean cadmium concentration measured in M. expansa was 21.5 ± 19.2 mg kg⁻¹ dry weight, which was 31 and 1.5 times higher than levels determined in the muscle and kidney of the host, respectively, but 0.9 times lower than levels determined in the liver of host. Sheeps with M. expansa infection always had higher cadmium concentrations in the tissues (with the exception of the blood) than their uninfected conspecifics.     

Oxidative enzymes in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

The activities of phenolase, peroxidase, cytochrome oxidase, catalase and superoxide dismutase, as well as the levels of lipid peroxides, were measured in plerocercoids of S. solidus taken from the body cavity of the fish (unactivated) and in plerocercoids which had been cultured in vitro, either under air, or under 95% N₂, 5% CO₂. When cultured anaerobically, the activities of phenolase, peroxidase and cytochrome oxidase all increased dramatically. Aerobically, only phenolase activity increased. Lipid peroxide levels and superoxide dismutase activity was similar at all stages and catalase could not be detected. It is suggested that the increased activity of oxidative enzymes in anaerobically cultured worms is an attempt to compensate for the reduced environmental pO₂.     

Cytokinin oxidase/dehydrogenase overexpression modifies antioxidant defense against heat,drought and their combination in Nicotiana tabacum plants

Cytokinins (CKs) as well as the antioxidant enzyme system (AES) play important roles in plant stress responses. The expression and activity of antioxidant enzymes (AE) were determined in drought, heat and combination of both stresses, comparing the response of tobacco plants overexpressing the main cytokinin degrading enzyme, cytokinin oxidase/dehydrogenase, under the control of root-specific WRKY6 promoter (W6:CKX1 plants) or constitutive promoter (35S:CKX1 plants) and the corresponding wild-type (WT). Expression levels as well as activities of cytosolic ascorbate peroxidase, catalase 3, and cytosolic superoxide dismutase were low under optimal conditions and increased after heat and combined stress in all genotypes. Unlike catalase 3, two other peroxisomal enzymes, catalase 1 and catalase 2, were transcribed extensively under control conditions. Heat stress, in contrast to drought or combined stress, increased catalase 1 and reduced catalase 2 expression in WT and W6:CKX1 plants. In 35S:CKX1, catalase 1 expression was enhanced by heat or drought, but not under combined stress conditions. Mitochondrial superoxide dismutase expression was generally higher in 35S:CKX1 plants than in WT. Genes encoding for chloroplastic AEs, stromatal ascorbate peroxidase, thylakoidal ascorbate peroxidase and chloroplastic superoxide dismutase, were strongly transcribed under control conditions. All stresses down-regulated their expression in WT and W6:CKX1, whereas more stress-tolerant 35S:CKX1 plants maintained high expression during drought and heat. The achieved data show that the effect of down-regulation of CK levels on AES may be mediated by altered habit, resulting in improved stress tolerance, which is associated with diminished stress impact on photosynthesis, and changes in source/sink relations.     

The susceptibility of strains of Mycobacterium tuberculosis to catalase-mediated peroxidative killing

At low pH and with continuous low concentrations of hydrogen peroxide generated in situ, catalase was able to replace peroxidase in the peroxidase/hydrogen peroxide/iodide microbicidal system. The system was effective against Escherichia coli and Mycobacterium tuberculosis. Iodide could not be replaced by chloride. The system was effective in lactate buffer, but not in citrate/phosphate buffer. Strains of M. tuberculosis with high and low virulence were equally susceptible. The observations are discussed in the context of an involvement of host-cell catalase in a possible intracellular killing mechanism against M. tuberculosis.     

Characteristics of a Urate-Degrading Diamine Oxidase-Peroxidase Enzyme System in Soybean Radicles

The cadaverine content of soybean radicles showed a maximumpeak 3–4 days after planting. The variation coincidedwith radicle uricase activity during seed germination. The uricase activity could not be fractionate when the bufferpH for the extraction was at 6.0. The addition of 1 M KCl orNaCl to the buffer allowed the extraction of the uricase activity,but an addition of 1 M MgCl₂ or BaCl₂ inhibited this enzyme'sactivity. The urate-degrading enzyme system was purified 248-fold permilligram of protein from soybean radicles. The respective K_mvalues of the diamine oxidase activity for cadaverine and ofthe urate-degrading activity for hydrogen peroxide and uratewere 1.25, 2.93 and 50.3 µM. Analysis by gel electrophoresisof the partially purified enzyme fraction revealed that theurate-degrading enzyme system consisted of a peroxidase thatdegrades urate with hydrogen peroxide and a diamine oxidasethat releases hydrogen peroxide. These data are evidence that a urate-degrading diamine oxidaseand peroxidase system exists in soybean radicles and that thereaction rate of urate-degradation is controlled by the concentrationof cadaverine. (Received November 28, 1984; Accepted April 8, 1985)     

南方根结线虫对不同砧木嫁接番茄苗活性氧清除系统的影响

采用盆栽人工接种方法,对番茄嫁接苗进行了抗性评价,研究了番茄嫁接苗叶片中抗氧化酶活性和活性氧代谢的动态变化。结果表明,接种南方根结线虫(J2)后,砧木嫁接苗表现为高抗,自根嫁接苗为高感。通过嫁接换根,与自根嫁接苗相比,砧木嫁接苗明显提高了接穗叶片的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性,降低了超氧阴离子(O.2-)产生速率以及过氧化氢(H2O2)和丙二醛(MDA)含量。表明番茄植株体内的活性氧水平和抗氧化酶活性的高低与其抗根结线虫的能力密切相关,较低的活性氧水平和较高的抗氧化酶活性有利于减轻对膜系统的伤害,提高番茄植株的抗根结线虫能力。     

Identification of Genetically Induced Lesions and the Sites of Action of Inhibitors Affecting Photorespiration by Simple Tests on Leaf Discs

Turner, J. C. and Hall, N. P. 1988. Identification of geneticallyinduced lesions and sites of action of inhibitors affectingphotorespiration by simple tests on leaf discs.—J. exp,Bot. 39: 345-351. Six photorespiratory mutants of barley deficient in catalaseand two mutants lacking phospho-glycollate phosphatase wereidentified by a novel screening method using leaf discs. Leaf discs were punched directly into an appropriate bufferedreagent in which the enzymes diffused from the cut edges ofthe discs causing a change in the colour reagents. The reactionswere observed from 15 min onwards depending on which enzymeactivity was being followed. Hundreds of plants can be screenedrapidly for major differences in enzyme activity. The methods depend on the formation of a red product in thereaction of hydrogen peroxide (H₂O₂) with 4-aminoantipyrenein the presence of peroxidase. To detect P-glycollatc phosphataseand glycollate oxidase, the product of the linked reactions,H₂O₂, was measured. For catalase, the disappearance of addedH₂O₂ is followed. By omitting peroxidase from the colour reagentmixture, peroxidase activity in leaf discs can be measured. The method was evaluated by applying it to existing enzyme deficientmutants of barley lacking P-glycollate phosphatase and catalase.Further mutant plants were detected by this method. The techniquecould also be used to screen for inhibitors of the glycollatepathway for use as herbicides. Key words: Phosphoglycollate phosphatase, glycollate oxidase, catalase, peroxidase, hydrogen peroxide     

Purification, partial characterization, and possible role of catalase in the bacterium Vitreoscilla

Vitreoscilla is a gram-negative bacterium that contains a unique bacterial hemoglobin that is relatively autoxidizable. It also contains a catalase whose primary function may be to remove hydrogen peroxide produced by this autoxidation. This enzyme was purified and partially characterized. It is a protein of 272,000 Da with a probable A2B2 subunit structure, in which the estimated molecular size of A is 68,000 Da and that of B, 64,000 Da, and an average of 1.6 molecules of protoheme IX per tetramer. The turnover number for its catalase activity was 27,000 s-1 and the Km for hydrogen peroxide was 16 mM. The peroxidase activity measured using o-dianisidine was 0.6% that of the catalase activity. Cyanide, which inhibited both catalase and peroxidase activities, bound the heme in a noncooperative manner. Azide inhibited the catalase activity but stimulated the peroxidase activity. An apparent compound II was formed by the reaction of the enzyme with ethyl hydrogen peroxide. The enzyme was reducible by dithionite, and the ferrous enzyme reacted with CO. The cellular content of Vitreoscilla hemoglobin varies during the growth cycle and in cells grown under different conditions, but the ratio of hemoglobin to catalase activity remained relatively constant, indicating possible coordinated biosynthesis and supporting the putative role of Vitreoscilla catalase as a scavenger of peroxide generated by Vitreoscilla hemoglobin.     

THE KINETICS OF THE DECOMPOSITION OF PEROXIDE BY CATALASE

It has been shown that the experimental results obtained by Morgulis in a study of the decomposition of hydrogen peroxide by liver catalase at 20°C. and in the presence of an excess of a relatively high concentration of peroxide are quantitatively accounted for by the following mechanisms. 1. The rate of formation of oxygen is independent of the peroxide concentration provided this is greater than about 0.10 M. 2. The rate of decomposition of the peroxide is proportional at any time to the concentration of catalase present. 3. The catalase undergoes spontaneous monomolecular decomposition during the reaction. This inactivation is independent of the concentration of catalase and inversely proportional to the original concentration of peroxide up to 0.4 M. In very high concentrations of peroxide the inactivation rate increases. 4. The following equation can be derived from the above assumptions and has been found to fit the experiments accurately. See PDF for Equation in which x is the amount of oxygen liberated at the time t, A is the total amount of oxygen liberated (not the total amount available), and K is the inactivation constant of the enzyme.     

Oxidase Reaction of the Hybrid Mn-Peroxidase of the Fungus <Emphasis Type="Italic">Panus tigrinus</Emphasis> 8/18

The hybrid Mn-peroxidase of the fungus Panus tigrinus 8/18 oxidized NADH in the absence of hydrogen peroxide, this being accompanied by the consumption of oxygen. The reaction of NADH oxidation started after a period of induction and completely depended on the presence of Mn(II). The reaction was inhibited in the presence of catalase and super-oxide dismutase. Oxidation of NADH by the enzyme or by manganese(III)acetate was accompanied by the production of hydrogen peroxide and superoxide radicals. In the presence of NADH, the enzyme was transformed into a catalytically inactive oxidized form (compound III), and the latter was inactivated with bleaching of the heme. The substrate of the hybrid Mn-peroxidase (Mn(II)) reduced compound III to yield the native form of the enzyme and prevented its inactivation. It is assumed that the hybrid Mn-peroxidase used the formed hydrogen peroxide in the usual peroxidase reaction to produce Mn(III), which was involved in the formation of hydrogen peroxide and thus accelerated the peroxidase reaction. The reaction of NADH oxidation is a peroxidase reaction and the consumption of oxygen is due to its interaction with the products of NADH oxidation. The role of Mn(II) in the oxidation of NADH consisted in the production of hydrogen peroxide and the protection of the enzyme from inactivation.__________Translated from Biokhimiya, Vol. 70, No. 4, 2005, pp. 568–574.Original Russian Text Copyright © 2005 by Lisov, Leontievsky, Golovleva.