A more sensitive modification of the catalase assay with the Clark oxygen electrode : Application to the kinetic study of the pea leaf enzyme

A modification of the method of catalase determination by means of the Clark oxygen electrode is described. The assay is based on measurement of the initial rate at which oxygen is released by catalase in an oxygen-free buffer. Displacement of oxygen was brought about by flushing with nitrogen, and the substrate used was hydrogen peroxide at a 33.5 m final concentration. The method is rapid and can be used with crude catalase preparations. Its sensitivity is at least 20 times higher than that of previous methods; it has an interval of measurable activity of about 0.01–8.4 μmol of O₂/min and, therefore, is applicable to an 840-fold range of catalase concentrations. This modification was applied to the kinetic study of crude extracts of pea leaf catalase. An apparent K_m of 0.190 was calculated.     

Catalase activity is modulated by calcium and calmodulin in detached mature leaves of sweet potato

Catalase (CAT) functions as one of the key enzymes in the scavenging of reactive oxygen species and affects the H₂O₂ homeostasis in plants. In sweet potato, a major catalase isoform was detected, and total catalase activity showed the highest level in mature leaves (L3) compared to immature (L1) and completely yellow, senescent leaves (L5). The major catalase isoform as well as total enzymatic activity were strongly suppressed by ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). This inhibition could be specifically and significantly mitigated in mature L3 leaves by exogenous CaCl₂, but not MgCl₂ or CoCl₂. EGTA also inhibited the activity of the catalase isoform in vitro. Furthermore, chlorpromazine (CPZ), a calmodulin (CAM) inhibitor, drastically suppressed the major catalase isoform as well as total enzymatic activity, and this suppression was alleviated by exogenous sweet potato calmodulin (SPCAM) fusion protein in L3 leaves. CPZ also inhibited the activity of the catalase isoform in vitro. Protein blot hybridization showed that both anti-catalase SPCAT1 and anti-calmodulin SPCAM antibodies detect a band at the same position, which corresponds to the activity of the major catalase isoform from unboiled, but not boiled crude protein extract of L3 leaves. An inverse correlation between the major catalase isoform/total enzymatic activity and the H₂O₂ level was also observed. These data suggest that sweet potato CAT activity is modulated by CaCl₂ and SPCAM, and plays an important role in H₂O₂ homeostasis in mature leaves. Association of SPCAM with the major CAT isoform is required and regulates the in-gel CAT activity band.     

A partial characterization of the cyclic nucleotide phosphodiesterases of Drosophila melanogaster

The cyclic nucleotide phosphodiesterases in crude homogenate, soluble material, and particulate preparations of adult Drosophila melanogaster flies, hydrolyze cyclic AMP with nonlinear kinetics. Cyclic GMP is hydrolyzed by the phosphodiesterases in crude homogenate and soluble material with linear kinetics. Physical separation techniques of gel filtration, velocity sedimentation, and ion-exchange chromatography reveal that Drosophila soluble fraction contains two major forms of cyclic nucleotide phosphodiesterase. Form I hydrolyzes both cyclic AMP and cyclic GMP. Inhibition experiments suggest that the hydrolysis of both cyclic nucleotides by Form I occurs at a single active site. The K_m's for hydrolysis of both substrates are about 4 μm. This form has a molecular weight of about 168,000 as estimated by gel nitration. Form II cyclic nucleotide phosphodiesterase is specific for cyclic AMP as substrate. Gel filtration indicates that this form has a molecular weight of about 68,000. The K_m for cyclic AMP is about 2 μm.     

Quantification of hydrogen peroxide in plant extracts by the chemiluminescence reaction with luminol

The chemiluminescence of luminol (3-aminophthalhydrazide) with H₂O₂ has been used to quantify endogenous amounts of H₂O₂ in plant tissues. The reaction is linear over at least three orders of magnitude between 10^?5 and 10^?2M H₂O₂. Interference by coloured compounds in the crude extract is calibrated by a purification step with Dowex AG 1-X8. The extract is calibrated with an internal H₂O₂ standard, and the specificity verified by H₂O₂ purging with catalase. The minimum delectability for H₂O₂ of this assay is at least 1 ng, corresponding to 0.1–1 g fresh material. Data are presented for the levels of H₂O₂ in potatoes after treatment with oxygen and ethylene, in tomatoes before and after ripening and in untreated germinating castor beans as well as in beans treated with aminotriazol to inhibit catalase activity. Though data using the titanium test are generally confirmed, the method presented here has the advantage of higher sensitivity and specificity.     

Spatial organization of catalase proteins

The three-dimensional structure of the heme-containing fungal catalase fromPenicillium vitale (m.m. 2,80,000) has been studied by X-ray analysis at 2.0 A resolution. The molecule is tetramer, each subunit contains 670 aminoacid residues identified to construct “X-ray” primary structure. The subunit is built of three compact domains and their connections. The first domain of about 350 residues contains aβ-barrel flanked by helices, the second domain of 70 residues is formed by four helices and the third one is composed of 150 residues and is topologically similar to flavodoxin. The active site including heme is deeply buried near theβ-barrel. A comparison of the structure of catalase fromPenicillium vitale with that of beef liver catalase revealed very close structural homology of the first and the second domain, but the third domain is entirely absent in beef liver catalase. A catalase from thermophillic bacteriaThermus thermophilus (m.m. 2,10,000) has been first isolated, crystallized and studied by X-ray analysis. Crystals are cubic, space group is P2₁3, a = 133.4 Å. The molecule is a hexamer with trigonal symmetry 32. The electron density map at 3 Å resolution made it possible to trace the polypeptide chain. The main structural motif is formed by four near parallel helices. There is no heme inThermus thermophilus catalase, the active site is between the four helices and contains two manganese ions.     

The reaction of cysteine with ceruloplasmin copper

The kinetics of decay in absorbance at 610 nm in the reaction of cysteine with ceruloplasmin was biphasic under anaerobic conditions. Admission of oxygen to the bleached ceruloplasmin restored the blue color to about 75 % of the original value. However, under aerobic or anaerobic conditions an initial bleaching corresponded to a 25 % decrease in blue color. This change was irreversible and remained after removal of excess cysteine from the reaction mixture by dialysis. There was no correlation between transient and steady-state kinetic parameters. Circular dichroism measurements showed a characteristic reduction in the negative band at 450 nm, which is specific for type 1b copper. Isolation and further studies on cysteine-modified ceruloplasmin with a lower A₆₁₀/A₂₈₀ ratio showed < 10% reduction in enzyme activity toward p-phenylenediamine and o-dianisidine. Evidence is also presented that ceruloplasmin catalyzes the oxidation of cysteine with a one-electron reduction of oxygen and the formation of superoxide ion, which is then converted to H₂O₂ by ceruloplasmin. The effect of superoxide dismutase and catalase also confirms the presence of superoxide and H₂O₂. In sum, these data show that a permanent reduction of type 1b copper occurred when cysteine was used as a substrate. We conclude that there is a single electron transfer from cysteine directly to oxygen using one specific copper of ceruloplasmin, type 1b.     

Protection of Bacillus larvae from Oxygen Toxicity with Emphasis on the Role of Catalase

Sporulation of Bacillus larvae NRRL B-3650 occurred only at aeration rates lower than those used for cultivation of most Bacillus species. One possible explanation for the requirement for a low level of aeration in B. larvae is that toxic forms of oxygen such as H₂O₂ and superoxide are involved. The superoxide dismutase levels of strain B-3650 were similar to those of Bacillus subtilis 168 during sporulation, and no NADH peroxidase was present. Catalase activity was absent during exponential growth and first appeared near the start of the stationary phase. The catalase activity was 2,700 times less than that in B. subtilis 168 at the same stage of development. Therefore, the relative deficiency of catalase (and NADH peroxidase) might be the cause of the apparent O₂ toxicity. It was postulated that B. larvae might accumulate H₂O₂ in the medium and exhibit more than normal sensitivity to H₂O₂. Experimental results did not verify either postulate, but the possibilities of intracellular accumulation of H₂O₂ and unusual sensitivity to endogenous H₂O₂ were not excluded. The catalase present in early-stationary-phase cells was soluble, heat labile, and inhibited by cyanide, azide, and hydroxylamine. An increase in catalase activity also occurred at the time of appearance of refractile spores in both B. larvae NRRL B-3650 and B. subtilis 168. The level of catalase activity in strain B-3650 was 5,400 times less than that in B. subtilis 168 at this stage. In B. larvae, this second increase occurred primarily within the developing endospore. The activity in spore extracts was particulate, heat stable, and inhibited by hydroxylamine but not by azide or cyanide. Synthesis of catalase in B. larvae was unaffected by H₂O₂, O₂, or glucose.     

Hepatic microsomal ethanol-oxidizing system: solubilization, isolation, and characterization

The solubilization and subsequent separation of the hepatic microsomal ethanol-oxidizing system from alcohol dehydrogenase and catalase activities by DEAE-cellulose column chromatography is described. Absence of alcohol dehydrogenase in the column eluates exhibiting microsomal ethanol-oxidizing system activity was demonstrated by the failure of NAD⁺ to promote ethanol oxidation at pH 9.6. Differentiation of the microsomal ethanol-oxidizing system from alcohol dehydrogenase was further shown by the apparent K_m for ethanol (7.2 mm, insensitivity of the microsomal ethanol-oxidizing system to the alcohol dehydrogenase inhibitor pyrazole (0.1 mm) and by the failure of added alcohol dehydrogenase to increase the ethanol oxidation. Absence of catalatic activity in these fractions was demonstrated by spectrophotometric and polarographic assay. Differentiation of the microsomal ethanol-oxidizing system from the peroxidatic activity of catalase was shown by the apparent K_m for oxygen (8.3 μm), insensitivity of the microsomal ethanol-oxidizing system to the catalase inhibitors azide and cyanide, and by the lack of a H₂O₂-generating system (glucose-glucose oxidase) to sustain ethanol oxidation in the eluates. The oxidation of ethanol to acetaldehyde by the alcohol dehydrogenase- and catalase-free fractions required NADPH and oxygen and was inhibited by CO. The column eluates showing microsomal ethanol-oxidizing system activity contained cytochrome P-450, NADPH-cytochrome c reductase, and phospholipids and also metabolized aminopyrine, benzphetamine, and aniline.     

An inhibitor of catalase induced by cold in chilling-sensitive plants

An inhibitor of catalase accumulated when leaves of chilling-sensitive species were stored in the dark at 0°C. The inhibitor could be removed from crude extracts by passing them through a column of Sephadex G-25. After this treatment, the catalase activity of extracts of chilled tissues was found to be equal to that of extracts from unchilled leaves. When chilled tissues were incubated at 20°C, the inhibitor of catalase was lost, unless the tissues had been irreversibly damaged. It specifically inhibited plant catalase, and had no effect on mammalian catalase, plant malic dehydrogenase, or plant superoxide dismutase.

Despite the presence of catalase inhibitor in extracts of chilled plants, no increase in the level of H₂O₂ in chilled tissues was found, suggesting either that the inhibitor is compartmentalized and not in contact with catalase in vivo, or that the level of H₂O₂ is controlled by means other than through catalase activity. Plant tissues normally contain H₂O₂ which is destroyed by catalase when they are damaged. After chilling, H₂O₂ leaking from already injured cells would not be so readily removed by the inhibited catalase, and could contribute to further injury by acting as a source of free radical oxidants.

     

Sensitive method for determination of peroxidase activity in tissue by means of coupled oxidation reaction

A simple colorimetric one-step method for determination of peroxidase activity in tissue is described. The method utilizes enzymically released H₂O₂ from glucose oxidation and o-dianisidine as hydrogen donor. The sensitivity of the method is at least ten times greater than existing methods. The influence of H₂O₂ concentration, buffer composition, and catalase interference is studied and discussed. An alternative fluorometric modification is briefly described.     

Inorganic Polyphosphates Regulate Hexokinase Activity and Reactive Oxygen Species Generation in Mitochondria of Rhipicephalus (Boophilus) microplus Embryo

The physiological roles of polyphosphates (poly P) recently found in arthropod mitochondria remain obscure. Here, the possible involvement of poly P with reactive oxygen species generation in mitochondria of Rhipicephalus microplus embryos was investigated. Mitochondrial hexokinase and scavenger antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione reductase were assayed during embryogenesis of R. microplus. The influence of poly P₃ and poly P₁₅ were analyzed during the period of higher enzymatic activity during embryogenesis. Both poly Ps inhibited hexokinase activity by up to 90% and, interestingly, the mitochondrial membrane exopolyphosphatase activity was stimulated by the hexokinase reaction product, glucose-6-phosphate. Poly P increased hydrogen peroxide generation in mitochondria in a situation where mitochondrial hexokinase is also active. The superoxide dismutase, catalase and glutathione reductase activities were higher during embryo cellularization, at the end of embryogenesis and during embryo segmentation, respectively. All of the enzymes were stimulated by poly P₃. However, superoxide dismutase was not affected by poly P₁₅, catalase activity was stimulated only at high concentrations and glutathione reductase was the only enzyme that was stimulated in the same way by both poly Ps. Altogether, our results indicate that inorganic polyphosphate and mitochondrial membrane exopolyphosphatase regulation can be correlated with the generation of reactive oxygen species in the mitochondria of R. microplus embryos.     

Evidence for H2O2 as the product of reduction of oxygen by alternative oxidase in mitochondria from potato tubers

Oxygen consumption by alternative oxidase (AOX), present in mitochondria of many angiosperms, is known to be cyanide-resistant in contrast to cytochrome oxidase. Its activity in potato tuber (Solanum tuberosum L.) was induced following chilling treatment at 4 °C. About half of the total O₂ consumption of succinate oxidation in such mitochondria was found to be sensitive to SHAM, a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive oxygen consumption by nearly half, and addition at the end of the reaction released nearly half of the consumed oxygen by AOX, both typical of catalase action on H₂O₂. These findings with catalase suggest that the product of reduction of AOX is H₂O₂ and not H₂O, as previously surmised. In potatoes subjected to chill stress (4 °C) for periods of 3, 5 and ?8 days the activity of AOX in mitochondria increased progressively with a corresponding increase in the AOX protein detected by immunoblot of the protein.     

THE DECOMPOSITION OF HYDROGEN PEROXIDE BY LIVER CATALASE

1. The velocity of decomposition of hydrogen peroxide by catalase as a function of (a) concentration of catalase, (b) concentration of hydrogen peroxide, (c) hydrogen ion concentration, (d) temperature has been studied in an attempt to correlate these variables as far as possible. It is concluded that the reaction involves primarily adsorption of hydrogen peroxide at the catalase surface. 2. The decomposition of hydrogen peroxide by catalase is regarded as involving two reactions, namely, the catalytic decomposition of hydrogen peroxide, which is a maximum at the optimum pH 6.8 to 7.0, and the "induced inactivation" of catalase by the "nascent" oxygen produced by the hydrogen peroxide and still adhering to the catalase surface. This differs from the more generally accepted view, namely that the induced inactivation is due to the H₂O₂ itself. On the basis of the above view, a new interpretation is given to the equation of Yamasaki and the connection between the equations of Yamasaki and of Northrop is pointed out. It is shown that the velocity of induced inactivation is a minimum at the pH which is optimal for the decomposition of hydrogen peroxide. 3. The critical increment of the catalytic decomposition of hydrogen peroxide by catalase is of the order 3000 calories. The critical increment of induced inactivation is low in dilute hydrogen peroxide solutions but increases to a value of 30,000 calories in concentrated solutions of peroxide.     

Superoxide dismutase and the oxygen enhancement of radiation lethality

Escherichia coli B were more susceptible to radiation lethality and showed a greater oxygen enhancement ratio when exposed in dilute suspension (1 × 10⁵ cells/ml) than when exposed in dense suspensions (1 × 10⁹ cells/ml). The oxygen enhancement, seen with dilute suspensions, was diminished by superoxide dismutase, catalase, mannitol, or histidine. Heat-denatured superoxide dismutase was without effect. The results are interpreted as indicating a role for O₂^? plus H₂O₂ in the oxygen enhancement of radiation lethality, and a scheme is proposed which is consistent with the observations.     

Apparent Catalase Synthesis in Sunflower Cotyledons during the Change in Microbody Function: A Mathematical Approach for the Quantitative Evaluation of Density-labeling Data

Density-labeling with 10 mm K¹⁵NO₃/70% ²H₂O has been used to investigate catalase synthesis in different developmental stages of sunflower (Helianthus annuus L.) cotyledons. A mathematical approach is introduced for the quantitative evaluation of the density-labeling data. The method allows, in the presence of preexisting enzyme activity, calculation of this synthesized activity (apparent enzyme synthesis) which results from the balance between actual enzyme synthesis and the degradation of newly synthesized enzyme at a given time. During greening of the cotyledons, when the catalase activity declines and the population of leaf peroxisomes is formed, the apparent catalase synthesis is lower than, or at best equal to, that occurring during a developmental stage when the leaf peroxisome population is established and catalase synthesis and degradation of total catalase are in equilibrium. This result suggests a formation, in fatty cotyledons, of the leaf peroxisomes by transformation of the glyoxysomes rather than by de novo synthesis.     

Effect of oxygen on galactose oxidase synthesis and secretion inDactylium dendroides

Dactylium dendroides mycelia exposed to different oxygen tensions secreted galactose oxidase in proportion to the pO₂. The intracellular levels of galactose oxidase, catalase, and superoxide dismutase increased 10.4-, 2.3-, and 2.1-fold, respectively, when the oxygen tension was raised from zero to 100%. Oxygen consumption was enhanced by increased partial pressure of O₂ and was higher than CO₂ production above 40% O₂. The results suggest that galactose oxidase could participate in an oxygen protection mechanism and/or as a microbicidal agent inD. dendroides.     

Increased spontaneous chemiluminescence from liver homogenates and isolated hepatocytes upon inhibition of o2− and h2o2 utilization

The intracellular steady-state concentrations of hydrogen peroxide or Superoxide anion were increased by inhibiting either catalase, glutathione peroxidase, or Superoxide dismutase activities. Catalase was inhibited with aminotriazole while glutathione peroxidase activity was blocked by eliminating reduced glutathione after addition of either iodoacetamide diethylmaleate or phorone. The concentration of aminotriazole that stimulated chemiluminescence in 50% (60 mM) was very similar to the K_i for catalase activity (70 mM). Cyanide, an inhibitor of both catalase and Superoxide dismutase, stimulated chemiluminescence in 50% at a concentration (0.15 mM) which is much closer from the K_i for Superoxide dismutase (0.25 mM) than from the K_i for catalase (15 μM). The Superoxide dismutase inhibitor diethyldithiocarbamate also increased chemiluminescence six- to ten-fold. Depletion of reduced glutathione stimulated spontaneous chemiluminescence when its concentration decreased below 4.5 μmol · g liver⁻¹. The results shown herein suggest that the changes in the intracellular steady-state concentration occurring after inhibition of any antioxidant enzyme are responsible for the increased spontaneous chemilumi-nescence. Spontaneous chemiluminescence from intact cells may be used as a noninvasive method for monitoring intracellular free radical metabolism.     

Cellular and intracellular localization of catalase and acid phosphatase in the midgut of Lumbricus terrestris L.: A cell fractionation study

• 1.1. Cellular and intracellular localization of catalase and acid phosphomonoesterase in the midgut of Lumbricus terrestris was studied by use of tissue fractionation.
• 2.2. At least 60–70% of the catalase resides in the chloragocyte cytosol and the remaining 30–40% resides in gut epithelium peroxisomes.
• 3.3. One of the main functions of the chloragocyte catalase is probably scavenging for H₂O₂ arising from the interaction between blood heme-protein and oxygen.
• 4.4. A simple method for the histochemical detection of cytosol catalase is proposed.
• 5.5. About 10% of the gut acid phosphatase resides in chloragocyte lysosomes. The chloragosomes contain no acid phosphatase.

     

Modification of catalase and MAPK in Vicia faba cultivated in soil with high natural radioactivity and treated with a static magnetic field

The effects of a static magnetic field (SMF) and high natural radioactivity (HR) on catalase and MAPK genes in Vicia faba were investigated. Soil samples with high natural radioactivity were collected from Ramsar in north Iran where the annual radiation absorbed dose from background radiation is higher than 20 mSv/year. The specific activity of the radionuclides of ²³²Th, ²³⁶Ra, and ⁴⁰K was measured using gamma spectrometry. The seeds were planted either in the soil with high natural radioactivity or in the control soils and were then exposed to a SMF of 30 mT for 8 days; 8 h/day. Levels of expression of catalase and MAPK genes, catalase activity and H₂O₂ content were evaluated. The results demonstrated significant differences in the expression of catalase and MAPK genes in SMF- and HR-treated plants compared to the controls. An increase in catalase activity was accompanied by increased expression of its gene and accumulation of H₂O₂. Relative expression of the MAPK gene in treated plants, however, was lower than those of the controls. The results suggest that the response of V. faba plants to SMF and HR may be mediated by modification of catalase and MAPK.     

Influence of dissolved oxygen on the nitrification kinetics in a circulating bed biofilm reactor

The influence of dissolved oxygen concentration on the nitrification kinetics was studied in the circulating bed reactor (CBR). The study was partly performed at laboratory scale with synthetic water, and partly at pilot scale with secondary effluent as feed water. The nitrification kinetics of the laboratory CBR as a function of the oxygen concentration can be described according to the half order and zero order rate equations of the diffusion-reaction model applied to porous catalysts. When oxygen was the rate limiting substrate, the nitrification rate was close to a half order function of the oxygen concentration. The average oxygen diffusion coefficient estimated by fitting the diffusion-reaction model to the experimental results was around 66% of the respective value in water. The experimental results showed that either the ammonia or the oxygen concentration could be limiting for the nitrification kinetics. The latter occurred for an oxygen to ammonia concentration ratio below 1.5–2 gO₂/gN-NH₄ ⁺ for both laboratory and pilot scale reactors. The volumetric oxygen mass transfer coefficient (k _L a) determined in the laboratory scale reactor was 0.017?s^?1 for a superficial air velocity of 0.02?m s^?1, and the one determined in the pilot scale reactor was 0.040?s^?1 for a superficial air velocity of 0.031?m?s^?1. The k _L a for the pilot scale reactor did not change significantly after biofilm development, compared to the value measured without biofilm.