Multiple catalases in Bacillus subtilis.

Vegetative cells of Bacillus subtilis in logarithmic growth phase produced one catalase, labeled catalase 1, with a nondenatured molecular weight of 205,000. As growth progressed, other activity bands with slower electrophoretic mobilities on polyacrylamide gels appeared, including a series of bands with a common nondenatured molecular weight of 261,000, collectively labeled catalase 2, and a minor band, with a molecular weight of 387,000, labeled catalase 3. Purified spores contained only catalase 2, and it was not produced in spo0A- or spo0F-containing mutants. Strains deficient in catalase 1 or catalase 2 or both were selected after mutagenesis. Sensitivities of the two main catalases to NaCN, NaN3, hydroxylamine, and temperature were similar, but the apparent Kms for H2O2 differed, being 36.6 and 64.4 mM, respectively, for catalase 1 and catalase 2. The levels of catalase 1 increased 15-fold during growth into stationary phase and could be increased 30-fold by the addition of H2O2 to the medium. Catalase 2, which was not affected by H2O2, appeared only after the cells had reached stationary phase, and the maximum levels were only half of the basal level of catalase 1.     

Production and some properties of catalase and superoxide dismutase from the anaerobe Bacteroides distasonis.

The catalase level of Bacteroides distasonis (ATCC 8503, type strain) varied with the amount of hemin supplied to the medium when the cells were grown in either a prereduced medium containing 0.5% peptone, 0.5% yeast extract, and 1% glucose or in a prereduced, defined heme-deficient medium. The effect of hemin on catalase production could not be duplicated by ferrous sulfate or ferrous ammonium citrate. Catalase activity reached peak values in late log phase, whereas superoxide dismutase specific activity remained constant throughout the culture growth cycle. The catalase was a nondialyzable, cyanide and azide-sensitive, heat-labile protein that coeluted with bovine erythrocyte catalase from Sepharose 6 B. Analysis of polyacrylamide gels stained for catalase activity and for heme showed a correspondence between the single catalytic activity band and one of three heme-protein bands. These data suggest a heme-protein of approximately 250,000 molecular weight. The superoxide dismutase was a cyanide-insensitive protein of approximately 40,000 molecular weight that migrated electrophoretically on acrylamide gels as a single band of activity.     

Characterization of pACK4, a mobilizable plasmid from Staphylococcus simulans biovar staphylolyticus

Staphylococcus simulans biovar staphylolyticus, the lysostaphin-producing organism, contains five plasmids designated pACK1–pACK5. pACK4 was found to be relaxable and to share sequence similarity with a number of well-characterized mobilizable plasmids from other staphylococci. All mobilizable staphylococcal plasmids characterized to date mediate resistance to various antibiotics, but pACK4 is unique because it contains no recognizable antibiotic resistance genes. pACK4 was found to contain an origin of transfer (oriT) region that shares inverted repeat regions and the same nic site as several other mobilizable staphylococcal plasmids. The presence of this conserved oriT region suggested that pACK4 might be mobilized in the presence of a conjugative plasmid. Filter mating studies revealed that pACK4 was mobilized by the conjugative plasmid pGO1. In addition, pACK4 was found to be virtually identical to the recently described plasmid pVGA from Staphylococcus aureus, except that pVGA contains an additional region (vgaA) that confers resistance to pleuromutilin, streptogramin A, and lincosamide. The high sequence similarity among pACK4, pVGA, and several previously described mobilizable staphylococcal plasmids suggests a common origin for these plasmids.     

Multiplicity of Antioxidant Enzyme Catalase in Mouse Liver Cells

Multiplicity of catalase activity has been observed in crude homogenates from the tissue and cell lines of mouse liver by ethanol/Triton X-100/heat treatment. The five enzymatically active catalase bands were designated as CAT1, CAT2, CAT3, CAT4, and CAT5 with a nondenatured molecular mass of 270kDa, 258kDa, 229kDa, 2lOkDa, or 197kDa, respectively. Cultured mouse liver cell lines, mouse liver tissue homogenate, and pure mouse liver catalase showed only one catalase band (CAT1) after ethanol/Triton X-100 treatment at 4°C for 72 hr. The same treatment but incubated at 37°C for 72 hr yielded three bands (CAT2, CAT4, CAT5) in normal cell line, only one band (CAT5) in MNNG-transformed and SV40-transformed cells, two bands (CAT1, CAT4) in mouse liver tissue homogenates, and two bands (CAT1, CAT3) in pure mouse liver catalase. These five catalase bands were further biochemically characterized. The CATl, CAT2, and CAT3 are sensitive to heat (68°C, 1 min), while CAT4 and CAT5 are rather heat resistant. The sensitivity to catalase inhibitors, such as aminotriazole, azide, or cyanide varies among the isoforms. Protease inhibitors could prevent the formation of CAT3 and CAT4, but not CAT5. Treatment with protease, however, removed all forms of catalase except CAT5. We conclude from this study that the appearance of different catalase bands is likely due to epigenetic modification of the protein, particularly proteolysis. The lowered catalase activity in transformed cells might also be attributable to the loss of two catalase isoforms.     

Response of Plant-Colonizing Pseudomonads to Hydrogen Peroxide

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

The lysostaphin endopeptidase resistance gene (epr) specifies modification of peptidoglycan cross bridges in Staphylococcus simulans and Staphylococcus aureus.

Staphylococcus simulans biovar staphylolyticus produces an extracellular glycylglycine endopeptidase (lysostaphin) that lyses other staphylococci by hydrolyzing the cross bridges in their cell wall peptidoglycans. The genes for endopeptidase (end) and endopeptidase resistance (epr) reside on plasmid pACK1. An 8.4-kb fragment containing end was cloned into shuttle vector pL150 and was then introduced into Staphylococcus aureus RN4220. The recombinant S. aureus cells produced endopeptidase and were resistant to lysis by the enzyme, which indicated that the cloned fragment also contained epr. Treatments to remove accessory wall polymers (proteins, teichoic acids, and lipoteichoic acids) did not change the endopeptidase sensitivity of walls from strains of S. simulans biovar staphylolyticus or of S. aureus with and without epr. Immunological analyses of various wall fractions showed that there were epitopes associated with endopeptidase resistance and that these epitopes were found only on the peptidoglycans of epr+ strains of both species. Treatment of purified peptidoglycans with endopeptidase confirmed that resistance or susceptibility of both species was a property of the peptidoglycan itself. A comparison of the chemical compositions of these peptidoglycans revealed that cross bridges in the epr+ cells contained more serine and fewer glycine residues than those of cells without epr. The presence of the 8.4-kb fragment from pACK1 also increased the susceptibility of both species to methicillin.     

Overexpression of catalase provides partial protection to transgenic mouse beta cells.

Pancreatic beta cells are sensitive to reactive oxygen species and this may play an important role in type 1 diabetes and during transplantation. Beta cells contain low levels of enzyme systems that protect against reactive oxygen species. The weakest link in their protection system is a deficiency in the ability to detoxify hydrogen peroxide by the enzymes glutathione peroxidase and catalase. We hypothesize that the deficit in the ability to dispose of reactive oxygen species is responsible for the unusual sensitivity of beta cells and that increasing protection will result in more resistant beta cells. To test these hypotheses we have produced transgenic mice with increased beta cell levels of catalase. Seven lines of catalase transgenic mice were produced using the insulin promoter to direct pancreatic beta cell specific expression. Catalase activity in islets from these mice was increased by as much as 50-fold. Northern blot analysis of several tissues indicated that overexpression was specific to the pancreatic islet. Catalase overexpression had no detrimental effects on islet function. To test whether increased catalase activity could protect the transgenic islets we exposed them to hydrogen peroxide, streptozocin, and interleukin-1beta. Fifty-fold overexpression of catalase produced marked protection of islet insulin secretion against hydrogen peroxide and significantly reduced the diabetogenic effect of streptozocin in vivo. However, catalase overexpression did not provide protection against interleukin-1beta toxicity and did not alter the effects of syngeneic and allogenic transplantation on islet insulin content. Our results indicate that in the pancreatic beta cell overexpression of catalase is protective against some beta cell toxins and is compatible with normal function.     

Thermal stability of catalases active in dormant saffron (Crocus sativus L.) corms

Catalase activity was detected in crude extract prepared from dormant saffron (Crocus sativus L.) corms. The activity was independent of pH in the range 6.0 – 11.0. Thermostability studies suggested the presence of three isoenzymes with transition temperatures of 30°C, 45°C and 60°C, respectively, as given by Arrhenius plots. When stained for catalase activity gel electropherograms of extract revealed 3 distinct bands with apparent molecular weight of 323,000, 295,000 and 268,000, respectively. Thus it appeared that at least three isoenzymes of catalase were present in dormant saffron corms.     

Plasmid-encoded lysostaphin endopeptidase resistance of Staphylococcus simulans biovar staphylolyticus

Staphylococcus simulans biovar staphylolyticus, the lysostaphin-producing organism, secretes a staphylolytic endopeptidase (EC 3.4.99.17) that is encoded on plasmid pACK1. Susceptibility of pACK1-cured strains to lysis by endopeptidase established that resistance to this enzyme is not an inherent property of the organism but rather is encoded on this dispensable plasmid. Furthermore, the enzyme is not an autolysin that is essential for cell wall synthesis because strains lacking the endopeptidase gene grew normally.     

Maintenance of higher H(2)O(2) levels, and its mechanism of action to induce growth in breast cancer cells: Important roles of bioactive catalase and PP2A

We assessed the catalase bioactivity and hydrogen peroxide (H(2)O(2)) production rate in human breast cancer (HBC) cell lines and compared these with normal human breast epithelial (HBE) cells. We observed that the bioactivity of catalase was decreased in HBC cells when compared with HBE cells. This was also accompanied by an increase in H(2)O(2) steady-state levels in HBC cells. Silencing the catalase gene led to a further increase in the steady-state level of H(2)O(2) which was also accompanied by an increase in growth rate of HBC cells. Catalase activity was up regulated on treatment with superoxide (O(2)(-)) scavengers such as pegylated SOD (PEG-SOD, indicating inhibition of catalase by the increased O(2)(-) produced by HBC cells. Transfection of either catalase or glutathione peroxidase to HBC cells decreased intracellular H(2)O(2) levels and led to apoptosis of these cells. The H(2)O(2) produced by HBC cells inhibited PP2A activity accompanied by increased phosphorylation of Akt and ERK1/2. The importance of catalase bioactivity in breast cancer was further confirmed as its bioactivity was also decreased in human breast cancer tissues when compared to normal breast tissues. We conclude that inhibition of catalase bioactivity by O(2)(-) leads to an increase in steady-state levels of H(2)O(2) in HBC cells, which in turn inhibits PP2A activity, leading to phosphorylation of ERK 1/2 and Akt and resulting in HBC cell proliferation.     

Combustion products of 1,3-butadiene inhibit catalase activity and induce expression of oxidative DNA damage repair enzymes in human bronchial epithelial cells

1,3-Butadiene, an important petrochemical, is commonly burned off when excess amounts need to be destroyed. This combustion process produces butadiene soot (BDS), which is composed of a complex mixture of polycyclic aromatic hydrocarbons in particulates ranging in size from <1 μm to 1 mm. An organic extract of BDS is both cytotoxic and genotoxic to normal human bronchial epithelial (NHBE) cells. Based on the oxidizing potential of BDS, we hypothesized that an organic extract of this particulate matter would (1) cause enzyme inactivation due to protein amino acid oxidation and (2) induce oxidative DNA damage in NHBE cells. Thus, our aims were to determine the effect of butadiene soot ethanol extract (BSEE) on both enzyme activity and the expression of proteins involved in the repair of oxidative DNA damage. Catalase was found to be sensitive to BDS as catalase activity was potently diminished in the presence of BSEE. Using Western analysis, both the alpha isoform of human 8-oxoguanine DNA glycosylase (α-hOGG1) and human apurinic/apyrimidinic endonuclease (APE-1) were shown to be significantly overexpressed as compared to untreated controls after exposure of NHBE cells to BSEE. Our results indicate that BSEE is capable of effectively inactivating the antioxidant enzyme catalase, presumably via oxidation of protein amino acids. The presence of oxidized biomolecules may partially explain the extranuclear fluorescence that is detected when NHBE cells are treated with an organic extract of BDS. Overexpression of both α-hOGG1 and APE-1 proteins following treatment of NHBE cells with BSEE suggests that this mixture causes oxidative DNA damage.     

Catalase activity during the recovery of heat-stressed Staphylococcus aureus MF-31.

Sublethal heating of Staphylococcus aureus produced little loss of catalase activity, but incubation of the injured cells in tryptic soy broth, with or without 10% NaCl added, produced an 85% decrease in catalase activity. Cells recovered in tryptic soy broth demonstrated increases in catalase levels after approximately 5 h, whereas in tryptic soy broth with 10% NaCl the levels remained low for at least 12 h. Thus, the loss of catalase activity during the recovery period was greater than during the heat treatment.     

Catalase activity during the recovery of heat-stressed Staphylococcus aureus MF-31

Sublethal heating of Staphylococcus aureus produced little loss of catalase activity, but incubation of the injured cells in tryptic soy broth, with or without 10% NaCl added, produced an 85% decrease in catalase activity. Cells recovered in tryptic soy broth demonstrated increases in catalase levels after approximately 5 h, whereas in tryptic soy broth with 10% NaCl the levels remained low for at least 12 h. Thus, the loss of catalase activity during the recovery period was greater than during the heat treatment.     

A study of the catalase monomer produced by lyophilization

Lyophilization of Dounce and Mourtzikos beef liver catalase (Prep. Biochem. 11 (1981) 501-523) under specified conditions produced conformationally altered but not completely denatured catalase monomer which retained both significant catalatic activity and peroxidatic activity towards ethanol. The same lyophilization procedure used with Sigma Co. catalase produced a mixture of conformationally altered catalase monomer and conformationally altered tetramer which showed still higher catalatic and peroxidatic activities; this was attributed to the presence of the altered tetramer. The catalase monomer obtained by the use of Dounce and Mourtzikos catalase is completely reducible by dithionite, as shown by the two-banded spectrum of the reduced material, but apparently retains enough of its native conformation to show some enzymatic activity, since the fully denatured monomer shows no catalatic or peroxidatic activity towards ethanol. The conformationally altered catalase tetramer, which shows more enzymatic activity than the monomer, evidently retains a higher proportion of its native conformation than the monomer, but still appears to be fully reducible with dithionite. Horseradish peroxidase after reduction with dithionite shows spectral bands at positions close to those of reduced lyophilized catalase, but the relative band heights and contours are different. A possible explanation for the observed differences in lyophilization products depending on the starting material (Sigma Co. catalase versus catalase of Dounce and Mourtzikos) is presented.     

Mechanisms of cellular resistance to hydrogen peroxide, hyperoxia, and 4-hydroxy-2-nonenal toxicity: the significance of increased catalase activity in H2O2-resistant fibroblasts.

An H2O2-resistant variant (OC14) of the HA1 Chinese hamster fibroblast cell line which demonstrates a 20-fold increase in catalase activity was utilized in the study of mechanisms responsible for cellular resistance to hydrogen peroxide, oxygen, and 4-hydroxy-2-nonenal toxicity. HA1 and OC14 cells were treated with 9 mM aminotriazole which resulted in a 60 to 80% reduction in catalase activity. Pretreatment with aminotriazole resulted in significant sensitization to the toxicity of 1-h exposures to exogenously applied H2O2, which was proportional to the reduction in catalase activity. Treatment with aminotriazole produced significant sensitization to the toxicity of 95% O2 after 45 h of O2 exposure but no sensitization to the toxicity of a 1-h exposure to 50 microM 4-hydroxy-2-nonenal. Inhibition of catalase activity by aminotriazole had no effect on the metabolism of 4-hydroxy-2-nonenal by either cell line tested. These results support the conclusion that in H2O2-resistant cells, catalase activity is a major determinant of cellular resistance to H2O2 toxicity, whereas catalase activity has a limited role in cellular resistance to an acute exposure to 95% O2 and is unrelated to cellular resistance to 4-hydroxy-2-nonenal.     

Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase

Hypochlorous acid (HOCl) produced via the enzyme myeloperoxidase is a major antibacterial oxidant produced by neutrophils, and Met residues are considered primary amino acid targets of HOCl damage via conversion to Met sulfoxide. Met sulfoxide can be repaired back to Met by methionine sulfoxide reductase (Msr). Catalase is an important antioxidant enzyme; we show it constitutes 4-5% of the total Helicobacter pylori protein levels. msr and katA strains were about 14- and 4-fold, respectively, more susceptible than the parent to killing by the neutrophil cell line HL-60 cells. Catalase activity of an msr strain was much more reduced by HOCl exposure than for the parental strain. Treatment of pure catalase with HOCl caused oxidation of specific MS-identified Met residues, as well as structural changes and activity loss depending on the oxidant dose. Treatment of catalase with HOCl at a level to limit structural perturbation (at a catalase/HOCl molar ratio of 1:60) resulted in oxidation of six identified Met residues. Msr repaired these residues in an in vitro reconstituted system, but no enzyme activity could be recovered. However, addition of GroEL to the Msr repair mixture significantly enhanced catalase activity recovery. Neutrophils produce large amounts of HOCl at inflammation sites, and bacterial catalase may be a prime target of the host inflammatory response; at high concentrations of HOCl (1:100), we observed loss of catalase secondary structure, oligomerization, and carbonylation. The same HOCl-sensitive Met residue oxidation targets in catalase were detected using chloramine-T as a milder oxidant.     

Aspergillus fumigatus catalases: cloning of an Aspergillus nidulans catalase B homologue and evidence for at least three catalases

The presence of catalases in the water soluble fractions of three Aspergillus fumigatus strains was investigated using non-denaturing and denaturing polyacrylamide gel electrophoresis and Western analysis. Using non-denaturing polyacrylamide gel electrophoresis and staining for catalase activity, three separate catalases were identified. An A. fumigatus catalase gene (catB) was cloned from genomic DNA using the Aspergillus niger catR gene as a probe. Polyclonal antibodies were raised to a glutathione S-transferase-CatB fusion product expressed in Escherichia coli. Western analysis indicated that, under denaturing conditions, the polyclonal antibody recognised a 90-kDa band and under non-denaturing conditions, two separate bands were identified. These results indicate that A. fumigatus in addition to CatB, produces at least two other catalases, one of which is similar in size to CatB. The polyclonal antibody was also used to observe catalase expression in mice, experimentally infected with A. fumigatus. Staining was observed heterogeneously throughout the fungal hyphae. This result indicates that catalase is produced by A. fumigatus during invasive aspergillosis.     

Factors affecting catalase expression in Pseudomonas aeruginosa biofilms and planktonic cells

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 microM as FeCl(3)) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.     

Alkaline phosphatase retained in HepG2 hepatocarcinoma cells vs. alkaline phosphatase released to culture medium: difference of aberrant glycosylation

Liver tissue is the source of 90% of serum alkaline phosphatase (AP). The serum levels and structures of tumor marker proteins change under many disease conditions as well as cancer. The study was aimed at determining the type of alkaline phosphatase (AP) present in HepG2 hepatocellular carcinoma cell line. Alkaline phosphatase rich extracts of healthy human liver, HepG2 hepatocarcinoma cells, as well as the condition medium of HepG2 cells were prepared by extraction with 40% n-butanol and 30-50% acetone precipitation, and subjected to various chromatographic procedures. Lectin affinity chromatography of the samples with concanavalin A-Sepharose 4B showed considerable differences in the elution patterns. Non-denaturing polyacrylamide gel electrophoresis of the culture medium yielded a relatively slow migrating band of activity that coincided with none of the three bands of activity produced by the normal liver extract, nor with the bands of the cell pellet extract. Inhibition patterns were established by measuring the enzyme activities in the presence of varying concentrations of L-phenylalanine, L-leucine, L-homoarginine, and levamisole. The APs from the cell line were neuraminidase sensitive. According to the results the main AP produced and released to the medium by HepG2 cell line is an aberrantly glycosylated tissue non-specific AP. In addition, the differences between the cell-pellet AP and the culture medium AP seemed to stem from different sugar moieties in their structures.