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PubMed1982

Effects of halide ions on porcine kidney catalase.

The inactivating effects of halide ions on porcine kidney catalase were investigated. It was found that the inactivation of catalase was dependent on incubation time with halides and on their concentrations: the addition of 2 M NaCl reduced the activity to 18% of the original level, whereas the same extent of inactivation was obtained in the presence of 0.1 M NaF. Removal of excess halide ions by dialysis resulted in good recovery of enzyme activity for all halides examined except for KI. Additions of halide ions to catalase solution caused subtle but evident alterations in the absorption and CD spectra. We could detect clear difference spectra between the salt-treated and native catalase solutions. These difference spectra showed halide concentration dependence with an evident isosbestic point. From these changes and also changes in CD spectra, we deduced that fluoride, chloride, and bromide ions can bind with heme iron of the catalase molecule as ligands to form stable catalase-halide complexes, but iodide ions showed a different reactivity with catalase from other halides and may cause gross alteration in the structure or conformation of catalase. Dissociation constants (Kd) were estimated to be 2.5, 0.23, and 26 M for chloride, fluoride, and bromide complexes with catalase, respectively, and there is no heme-heme interaction during formation of the catalase-halide complexes as estimated from the Hill plot.

Animals↗
PubMed1985

Interaction with deoxycholate of rat liver peroxisomal and cytosolic catalase.

Rat liver catalase was found to interact with deoxycholate (DOC). When purified, the peroxisomal catalase was precipitated at pH 6 in the presence of DOC, whereas in the peroxisomal extract (with DOC) it was unsedimentable at pH 6. The membrane fraction in the extract interacted with the catalase instead of DOC, and prevented the precipitation of catalase with DOC at pH 6. The peroxisomal catalase seemed to be easily modified by lysosomal protease during manipulation, and this proteolytic cleavage rendered the molecule able to interact with the membrane. On the other hand, the cytosolic catalase, both in the cytosol fraction and in the purified preparation, sedimented at pH 6 in the presence of DOC. The cytosolic catalase was far more resistant to proteolytic modification than the peroxisomal catalase. The molecule of peroxisomal catalase is assumed to have a site for recognizing the membrane, whereas such a structure may be absent in the cytosolic catalase or may not be easily exposed by proteolytic cleavage.

Animals↗
PubMed2000

Catalase deficiency in Staphylococcus aureus subsp. anaerobius is associated with natural loss-of-function mutations within the structural gene.

Degenerate oligonucleotide primers based on internal peptide sequences obtained by HPLC from purified Staphylococcus aureus catalase were used to locate the S. aureus and S. aureus subsp. anaerobius kat regions by PCR. Southern hybridization analysis with a probe derived from a 1.1 kb PCR-amplified fragment showed that a single copy of the putative catalase gene was present in the S. aureus and S. aureus subsp. anaerobius chromosome. The nucleotide sequence of S. aureus katA revealed a 1518 bp open reading frame for a protein with 505 amino acids and a predicted molecular mass of 58347 Da, whereas S. aureus subsp. anaerobius katB is 1368 nt long and encodes a polypeptide of 455 amino acids with a predicted molecular mass of 52 584 Da. These catalases are highly homologous to typical monofunctional catalases from prokaryotes. The active-site residues, proximal and distal haem-binding ligands and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in S. aureus KatA. Escherichia coli cells carrying cloned katA had a catalase activity approximately 1000 times that of untransformed E. coli, but no detectable increase in catalase activity was observed with E. coli carrying cloned katB. Northern blotting showed the presence of a kat-specific transcript in S. aureus subsp. anaerobius, suggesting that the lack of catalase activity in this bacterium is due to a post-transcriptional alteration. Compared to the nucleotide sequence of katA, katB showed a single base-pair deletion and six mis-sense mutations, and these alterations were present in three other S. aureus subsp. anaerobius strains analysed. The deletion, located at 1338 bp from the initiation codon, originates a shift of the nucleotide reading frame and is responsible for the premature translation termination at 1368 bp, generating a KatB polypeptide 50 amino acid residues shorter than KatA. Moreover, four of the mis-sense mutations present in katB lead to non-conservative amino acid replacements, the most significant being that located at residue 317 (Pro in KatA-->Ser in KatB) because the affected amino acid is involved in determining the proximal haem-binding site. Both the main alterations found in KatB (the deletion and the substitution in residue 317) seem to contribute to the lack of catalase activity in S. aureus subsp. anaerobius, as deduced from results obtained with chimeric catalase constructs.

Amino Acid Sequence↗
PubMed1998

Manipulation of catalase levels produces altered photosynthesis in transgenic tobacco plants.

Constructs containing the cDNAs encoding the primary leaf catalase in Nicotiana or subunit 1 of cottonseed (Gossypium hirsutum) catalase were introduced in the sense and antisense orientation into the Nicotiana tabacum genome. The N. tabacum leaf cDNA specifically overexpressed CAT-1, the high catalatic [corrected] form, activity. Antisense constructs reduced leaf catalase specific activities from 0.20 to 0.75 times those of wild type (WT), and overexpression constructs increased catalase specific activities from 1.25 to more than 2.0 times those of WT. The NADH-hydroxypyruvate reductase specific activity in transgenic plants was similar to that in WT. The effect of antisense constructs on photorespiration was studied in transgenic plants by measuring the CO2 compensation point (gamma) at a leaf temperature of 38 degrees C. A significant linear increase was observed in gamma with decreasing catalase (at 50% lower catalase activity gamma increased 39%). There was a significant temperature-dependent linear decrease in gamma in transgenic leaves with elevated catalase compared with WT leaves (at 50% higher catalase gamma decreased 17%). At 29 degrees C, gamma also decreased with increasing catalase in transgenic leaves compared with WT leaves, but the trend was not statistically significant. Rates of dark respiration were the same in WT and transgenic leaves. Thus, photorespiratory losses of CO2 were significantly reduced with increasing catalase activities at 38 degrees C, indicating that the stoichiometry of photorespiratory CO2 formation per glycolate oxidized normally increases at higher temperatures because of enhanced peroxidation.

Alcohol Oxidoreductases↗
PubMed1987

Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA.

A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313-317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.

Amino Acid Sequence↗
PubMed1977

Differentiation of catalases in Mycobacterium phlei on the basis of susceptibility to isoniazid: association with peroxidase and acquired resistance to isoniazid.

Mycobacterium phlei contains two catalase activities and a single peroxidase activity. The latter is associated with one of the catalases. The single catalase-peroxidase enzyme accounted for 75% of the total catalase activity and was lost upon acquisition of resistance to the antitubercular drug isoniazid (INH). Heat-treated (68 degrees C) wild-type cells showed similar decreases in catalase activity as well as complete loss of peroxidase activity. Catalase activity in the INH-resistant strain of M. phlei (Inh(r)) was unaffected by heating. The heat-sensitive catalase of the wild-type M. phlei was completely inhibited by 0.1 M INH, and Cu(2+) enhanced this inhibitory effect by 100-fold. No inhibition of activity was found with the heat-stable enzyme. Equivalent inhibition of catalase was also observed with nicotinic acid hydrazide and benzoic acid hydrazide. Peroxidase activity was also completely inhibited by any one of the three hydrazides, either INH, benzoic acid hydrazide, or nicotinic acid hydrazide at 10(-3) M. The presence of two catalase activities and the loss of one (catalase-peroxidase) on acquiring INH resistance or heating wild-type cells was confirmed by acrylamide gel electrophoresis of the cell-free extracts.

Bacterial Proteins↗
PubMed1986

Catalases, peroxidases, and superoxide dismutases in Mycobacterium leprae and other mycobacteria studied by crossed immunoelectrophoresis and polyacrylamide gel electrophoresis.

The five mycobacteria Mycobacterium lepraemurium, M. leprae, M. bovis BCG, M. smegmatis, and M. intracellulare were studied. Catalase and peroxidase activities were demonstrated in polyacrylamide and crossed immunoelectrophoresis gels for M. lepraemurium, M. intracellulare, and BCG, but not for M. leprae. Peroxidase and catalase activities were associated with the same precipitate line in crossed immunoelectrophoresis for M. lepraemurium, M. intracellulare, and BCG, showing that in these mycobacteria the two enzyme activities resided in the same molecule. M. smegmatis peroxidase and catalase activities were closely associated on polyacrylamide gel electrophoresis, but on the crossed immunoelectrophoresis catalase and peroxidase activities were associated with two different precipitate lines. Catalases without peroxidase activity were demonstrated in crossed immunoelectrophoresis and polyacrylamide gel electrophoresis in M. intracellulare and M. smegmatis. The catalase without peroxidase activity in M. intracellulare was heat resistant and therefore classified as an m-catalase. In M. smegmatis the catalase without peroxidase activity was only partially heat resistant. All of the catalases with peroxidase activity were heat-sensitive t-catalases. Superoxide dismutase activity in the crossed immunoelectrophoresis was associated with the M. leprae antigen no. 4 and with cross-reacting antigens in the other mycobacteria studied. Several superoxide dismutases were demonstrated in Mycobacterium duvalii. They were antigenically different from the other superoxide dismutases in this study, as shown by lack of reactivity with a monospecific antibody to M. lepraemurium superoxide dismutase. Molecular weights were estimated for all the enzymes in this study by sodium dodecyl sulfate-polyacrylamide gels.

Catalase↗
PubMed1998

Cloning and sequencing of a Candida albicans catalase gene and effects of disruption of this gene.

Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a single C. albicans-derived catalase gene, designated CAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicans ATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of C. albicans. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a CAT1-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation using URA3 as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of CAT1 with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of C. albicans.

Amino Acid Sequence↗
PubMed1990

Effect of superoxide dismutase and catalase, given separately, on myocardial "stunning".

Controversy persists regarding which oxygen metabolites are cytotoxic. Although the combination of superoxide dismutase (SOD) and catalase has been shown to attenuate postischemic myocardial dysfunction ("stunning"), it is unknown whether this beneficial effect is due to scavenging of O2-., H2O2, or both. Accordingly, 85 open-chest dogs underwent a 15-min occlusion of the left anterior descending coronary artery followed by 4 h of reperfusion. In phase A, dogs received an intravenous infusion of saline (group I), SOD (5 mg/kg, group II), catalase (12,000 U/kg, group III), or the combination of SOD and catalase (same doses, group IV). Recovery of regional myocardial function (assessed as systolic wall thickening) after reperfusion was significantly improved by the combination of SOD and catalase but not by SOD alone or catalase alone. To determine whether higher doses of enzymes are more effective, in phase B dogs received an intracoronary infusion of normal saline (group V), SOD in low dose (1.5 mg/kg, group VI), SOD in high dose (6.3 mg/kg plus 1.5 mg/kg iv, group VII), catalase in low dose (18,000 U/kg, group VIII), or catalase in high dose (240,000 U/kg plus 40,000 U/kg iv, group IX). Despite the fact that the local plasma levels of enzymes were considerably higher than those achieved in phase A, none of the treatments in phase B significantly enhanced recovery of contractile function. This study demonstrates that the combination of SOD and catalase is more effective than either enzyme alone in attenuating postischemic myocardial dysfunction and that increasing the doses of SOD or catalase does not provide additional protection. The results suggest that both O2-. and H2O2 contribute significantly to the pathogenesis of myocardial stunning after regional ischemia in the intact animal. Furthermore, the data imply that if SOD and catalase are to be used clinically to prevent postischemic dysfunction, protection may be achieved most effectively by combining the two enzymes.

Animals↗
PubMed2001

Blood catalase and haematocrit values in a breeding colony of Dutch-belted rabbits.

Rabbit seminal plasma catalase is much higher than in the semen of other mammals, and differences appear to be inherited. Because of the scarcity of information on rabbit blood catalase and haematocrit in Dutch-belted rabbits, an investigation of possible effects of gender, age and genetics on these variables was undertaken. There were 191 rabbits sampled at 2-3 months, 130 at 12 months and 61 at 18-24 months of age. There was no age effect on the haematocrit values and on blood catalase activity. At 12 months of age males had an average haematocrit value of 44% compared with 40% for females (P < 0.05). Corresponding average catalase values were 431 and 356 units/ml of blood (P < 0.05). Also catalase was measured in the semen and blood of 34 males, and males differed in both their blood and semen catalase activity (P < 0.05). The correlation between the two traits was r = 0.44. Heritability (h2) estimates, based on 231 rabbits were 0.40 for blood catalase activity, and 0.26 for haematocrit. The genetic correlation between the two variables was 0.83 (P < 0.05). These studies are consistent with the literature in that female rabbits have a slightly lower haematocrit value than males, and this is associated with a lower catalase activity. This appears to be the first report of a study that compares rabbit blood catalase in males and females of different ages. Preliminary evidence that differences may have a heritable basis is consistent with previous studies on rabbit semen catalase.

Age Factors↗
PubMed2006

Catalases are NAD(P)H-dependent tellurite reductases.

Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO(3)(2-)) to the less toxic, insoluble metal, tellurium (Te(o)), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical.

Amino Acid Sequence↗
PubMed1993

Cross-linked hemoglobin-superoxide dismutase-catalase scavenges oxygen-derived free radicals and prevents methemoglobin formation and iron release.

In this study, we prepared PolyHb-SOD-catalase (intermolecularly cross-linked hemoglobin, superoxide dismutase (SOD), and catalase). We found that PolyHb-SOD-catalase is effective in scavenging oxygen-derived free radicals. In the xanthine/xanthine oxidase system, the initial rate of cytochrome c reduction was 2.13 +/- 0.26 nmoles cyt. c/min for PolyHb alone. PolyHb- SOD-catalase reduced this to 0.56 +/- 0.08 nmoles cyt. c/min because of its ability to eliminate superoxide (O2-). Addition of PolyHb to 200 microM of hydrogen peroxide (H2O2), changed the H2O2 level slightly to 192 +/- 0.4 microM. Addition of PolyHb-SOD-catalase, on the other hand, lower the level to 41 +/- 0.3 microM. Results also show that both effects were dependent on the concentration of SOD-catalase cross-linked with hemoglobin. Oxidative challenge with H2O2 resulted in minimal changes in the absorbance spectra of PolyHb-SOD-catalase. With PolyHb, there were spectral changes reflecting the formation of methemoglobin and heme degradation. Furthermore, the amount of iron released, after incubation with 250 microM H2O2, was 6.8 +/- 1.8 micrograms/dl for PolyHb-SOD-catalase and 76.6 +/- 1.0 micrograms/dl for PolyHb. These results show that cross-linked SOD-catalase prevents oxidative reactions involving the hemoglobin component of PolyHb-SOD-catalase.

Catalase↗
PubMedDate not supplied

[Comparative kinetic characteristics of catalase of Penicillium species molds].

Extracellular catalases produced by fungi of the genus Penicillium: P. piceum, P. varians and P. kapuscinskii were purified by consecutive filtration of culture liquids. The maximum reaction rate of H2O2 decomposition, the Michaelis constants and specific catalytic activities of isolated catalases were determined. The operational stability was characterized by effective rate of catalase inactivation during enzymatic reaction (kin at 30 degrees C). The thermal stability was determined by the rate of enzyme thermal inactivation at 45 degrees C (k*[symbol: see text]H, s-1). Catalase from P. piceum displayed the maximum activity, which was higher than the activity of catalase from bovine liver. The operational stability of catalase from P. piceum was twofold to threefold higher than the stability of catalase from bovine liver. The physicochemical characteristics of catalases of fungi are better than the characteristics of catalase from bovine liver and intracellular catalase of yeast C. boidinii.

Animals↗
PubMed1975

Effect of injection of nuclear fraction from Rhodamine sarcoma on turnover of liver catalase.

1) When nuclear fraction prepared from Rhodamine sarcoma (sarcoma nuclear fraction) was injected into mice three times every 24 hr, the catalase activity of the liver decreased to one-third of the original activity. 2) By the injection of sarcoma nuclear fraction into mice, the catalase activity with the soluble fraction from homogenates of the liver decreased more significantly than that with the particulate fraction from them. 3) Immunological titration proved that the decrease of catalase activity in the liver of mice injected with sarcoma nuclear fraction was brought about by decrease in the amount of catalase protein. 4) In the mice, whose liver catalase activity had been irreversibly inhibited by injection of 3-amino-1,2,4-triazole, the initial rate for the restoration of the liver catalase activity was significantly showed by further injection of sarcoma nuclear fraction. 5) When the inhibitor of catalase biosynthesis, allylisopropylacetamide, was injected into mice, the activity level of the liver catalase decreased. The extent of decrease by the injection of the inhibitor was slightly lower than that by the injection with sarcoma nuclear fraction, which was almost the same as the extent of decrease by the injection of sarcoma nuclear fraction plus allylisopropylacetamide. 6) It is conceivable that the catalase biosynthesis in the liver was inhibited by the injection of sarcoma nuclear fraction in almost the same manner as by the injection of allylisopropylacetamide. However, it is not certain whether the degradation of liver catalase was slightly stimulated by the injection of sarcoma nuclear fraction.

Allylisopropylacetamide↗
PubMedDate not supplied

[Kinetic characteristics of extracellular catalase from Penicillium piceum F-648 and variants of fungi, adapted to hydrogen peroxide].

A comparative kinetic study of extracellular catalases produced by Penicillium piceum F-648 and their variants adapted to H2O2 was performed in culture liquid filtrates. The specific activity of catalase, the maximum rate of catalase-induced H2O2 degradation (Vmax),Vmax/KM ratio, and the catalase inactivation rate constant in the enzymatic reaction (kin, s-1) were estimated in phosphate buffer (pH 7.4) at 30 degrees C. The effective constant representing the rate of catalase thermal inactivation (kin*, s-1) was determined at 45 degrees C. In all samples, the specific activity and KM for catalase were maximum at a protein concentration in culture liquid filtrates of 2.5-3.5 x 10(-4) mg/ml. The effective constants describing the rate of H2O2 degradation (k, s-1) were similar to that observed in the initial culture. These values reflected a twofold decrease in catalase activity in culture liquid filtrates. We hypothesized that culture liquid filtrates contain two isoforms of extracellular catalase characterized by different activities and affinities for H2O2. Catalases from variants 5 and 3 with high and low affinities for H2O2, respectively, had a greater operational stability than the enzyme from the initial culture. The method of adaptive selection for H2O2 can be used to obtain fungal variants producing extracellular catalases with improved properties.

Adaptation, Physiological↗
PubMed2005

[The protective effect of recombinant adenovirus of catalase on oxidative damaged rat lens].

OBJECTIVES: To investigate the validity of catalase recombinant adenovirus on the treatment of oxidative cataract. METHODS: The coding sequence of catalase was cloned and the catalase recombinant adenovirus was constructed. The expression time course of catalase gene in rat lens infected by recombinant adenovirus was determined by Western blotting. Cultured rat lens were randomly divided into 3 groups: the control group, the group treated by hydrogen peroxide and the group treated by hydrogen peroxide combined with catalase recombinant adenovirus. The transparence and apoptosis ratio of lens on the time points of 6, 12, 18, 24 hours were determined by image analysis and double colour flowcytometry. RESULTS: The coding sequence of catalase was cloned and recombinant adenovirus was successfully constructed. The expression of catalase in cultured rat lens infected by recombinant adenovirus reached peak point on 9 hours post infection and maintained the level in the whole experiment period. The transparence of the lens in the group treated by hydrogen peroxide combined with catalase recombinant adenovirus was higher than that of group treated by hydrogen peroxide and lower than that of the control group on the time points of 6, 12, 18, 24 hours post infection. The differences among groups were statistically significant (P < 0.05). On the same time points, the apoptosis ratio of the group treated by hydrogen peroxide combined with catalase recombinant adenovirus was lower than that of the group treated by hydrogen peroxide and higher than the control group. The differences among groups were statistically significant (P < 0.05). CONCLUSION: The catalase recombinant adenovirus, which can inhibit the turbidity and cell apoptosis of lens caused by oxidant, may be used as the gene therapy of oxidative cataract.

Adenoviruses, Human↗
PubMed2006

Purification and characterization of a catalase from photosynthetic bacterium Rhodospirillum rubrum S1 grown under anaerobic conditions.

The photosynthetic bacterium, Rhodospirillum rubrum S1, when grown under anaerobic conditions, generated three different types of catalases. In this study, we purified and characterized the highest molecular weight catalase from the three catalases. The total specific catalase activity of the crude cell extracts was 88 U/mg. After the completion of the final purification step, the specific activity of the purified catalase was 1,256 U/mg. The purified catalase evidenced an estimated molecular mass of 318 kDa, consisting of four identical subunits, each of 79 kDa. The purified enzyme exhibited an apparent Km value of 30.4 mM and a Vmax of 2,564 U against hydrogen peroxide. The enzyme also exhibited a broad optimal pH (5.0-9.0), and remained stable over a broad temperature range (20 degrees C-60 degrees C). It maintained 90% activity against organic solvents (ethanol/chloroform) known hydroperoxidase inhibitors, and exhibited no detectable peroxidase activity. The catalase activity of the purified enzyme was reduced to 19% of full activity as the result of the administration of 10 mM 3-amino-1,2,4-triazole, a heme-containing catalase inhibitor. Sodium cyanide, sodium azide, and hydroxylamine, all of which are known heme protein inhibitors, inhibited catalase activity by 50% at concentrations of 11.5 microM, 0.52 microM, and 0.11 microM, respectively. In accordance with these findings, the enzyme was identified as a type of monofunctional catalase.

Amitrole↗
PubMed1991

Erythrocyte defense against hydrogen peroxide: preeminent importance of catalase.

To investigate the relative importance of catalase and glutathione in erythrocyte oxidant defense, human and mouse (normal and acatalasemic) erythrocytes were reversibly lysed and resealed in the presence of exogenous catalase or glutathione. This resulted in an increase in intracellular catalase activity or glutathione concentration in the resealed erythrocytes while normal cellular structure, hemoglobin concentration, cell volume, cellular deformability, and adenosine triphosphate concentration were maintained. Resealing alone had no effect on oxidant sensitivity. In human cells, a threefold increase in catalase activity resulted in the maintenance of glutathione levels in response to hydrogen peroxide (H2O2) challenge. Reconstitution of congenitally acatalasemic mouse erythrocytes, which were extremely sensitive to even micromolar concentrations of H2O2 with purified catalase resulted in complete protection against H2O2. Indeed, the catalase-reconstituted acatalasemic cells were less sensitive to H2O2-mediated damage than were normal, catalase-replete mouse cells. In contrast, alteration of the glutathione status of human and mouse (normal and acatalasemic) cells had no significant effect on oxidant sensitivity. Even a five-fold increase in intracellular glutathione concentration (greater than 30 micromoles glutathione per gram of hemoglobin) in normal or catalase-deficient (azide-treated or acatalasemic) red blood cells had no protective effect against H2O2-mediated lipid peroxidation or methemoglobin generation. Similarly, depletion of glutathione by 1-chloro-2,4-dinitrobenzene also had no effect on erythrocyte H2O2 sensitivity. These results suggest an important role for catalase in protection against H2O2-mediated damage at physiologic levels and that catalase is as at least as important as glutathione in cellular defense against H2O2.

Animals↗