Properties of residual catalase in the erythrocytes of Japanese-type acatalasemia

Summary In Japanese-type acatalasemia erythrocytes, the presence and properties of residual catalase were determined and compared with those of normal erythrocyte catalase. Residual catalase activity was proved by titration, active staining after polyacrylamide gel electrophoresis, and measurement of oxygen evolution. Residual catalase protein, demonstrated by double immunodiffusion, was similar to that of normal catalase. The properties of residual catalase activity were identical with those of normal catalase activity. It occurred as three fractions of equal specific activity by DEAE column chromatography. These observations suggest that Japanese-type acatalasemia contains residual catalase with properties similar to those of normal catalase.     

Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule per holomer and has an estimated molecular weight of 124,000 to 130,000. The catalase gene was cloned by screening a B. fragilis library for complementation of catalase activity in an Escherichia coli catalase mutant (katE katG) strain. The cloned gene, designated katB, encoded a catalase enzyme with electrophoretic mobility identical to that of the purified protein from the B. fragilis parental strain. The nucleotide sequence of katB revealed a 1,461-bp open reading frame for a protein with 486 amino acids and a predicted molecular weight of 55,905. This result was very close to the 60,000 Da determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified catalase and indicates that the native enzyme is composed of two identical subunits. The N-terminal amino acid sequence of the purified catalase obtained by Edman degradation confirmed that it is a product of katB. The amino acid sequence of KatB showed high similarity to Haemophilus influenzae HktE (71.6% identity, 66% nucleotide identity), as well as to gram-positive bacterial and mammalian catalases. No similarities to bacterial catalase-peroxidase-type enzymes were found. The active-site residues, proximal and distal hemebinding ligands, and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in B. fragilis KatB.     

Superoxide dismutase, catalase and nitrogenase activities of symbiotic Frankia (Alnus incana) in response to different oxygen tensions

Presence and activity of the enzymes superoxide dismutase (SOD) and catalase were studied in Frankia in symbiosis with Alnus incana (L.) Moench. Analysis on native PAGE gels indicated that symbiotic Frankia contained an FeSOD and catalase. The activity of the enzymes was in the same range as reported for cultured Frankia . Attempts to characterize SOD by western blots with antisera from Escherichia coli and Azotobacter vinelandii did not give clear-cut results with the antibodies used. Alnus incana plants were grown with the root system in 5, 10, 21 or 40% O₂ for up to 6 days. Nitrogenase activity, measured as ARA (acetylene reducing activity) dropped within 3 h when roots were exposed to low or high oxygen. At 40% O₂ ARA was almost completely lost while at 5 and 10% O₂ ARA decreased to 69 and 74% of the inital value, respectively, Nitrogenase activity recovered at ail oxygen tensions. Recovery rates resembled the continuous increase in ARA in plants continuosly kept at 21% O₂, and suggests that new vesicles with envelopes of appropriate thickness were formed. The ARA measurements confirm results from an earlier study where nitrogenase activity was measured as H₂ evolution. There was a tendency for increased SOD and catalase activities in Frankia from root systems exposed to 40% O₂ for 24 h but not earlier or later than this. When data from all experimental times were pooled. SOD activity increased significantly with increased oxygen tension whereas catalase activity decreased. Although ARA per plant varied with oxygen tension, there was no statistically significant correlation between ARA and SOD or between ARA and catalase. It seems that being linked to nitrogenase activity is only one role of SOD and catalase in this symbiotic Frankia .     

A novel analytical method to evaluate directly catalase activity of microorganisms and mammalian cells by ESR oximetry

Abstract

Electron spin resonance (ESR) oximetry technique was applied for analysis of catalase activity in the present study. Catalase activity was evaluated by measuring oxygen from the reaction between hydrogen peroxide (H₂O₂) and catalase-positive cells. It was demonstrated that the ESR spectra of spin-label probes, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO) and 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy (4-maleimido-TEMPO) in the presence of H₂O₂ were broadened with the concentrations of catalase. It was possible to make a calibration curve for catalase activity by peak widths of the spectra of each spin-label probe, which are broadened dependently on catalase concentrations. The broadened ESR spectra were also observed when the catalase-positive micro-organisms or the mammalian cells originally from circulating monocytes/macrophages were mixed with TEMPOL and H₂O₂. Meanwhile, catalase-negative micro-organisms caused no broadening change of ESR spectra. The present study indicates that it is possible to evaluate directly the catalase activity of various micro-organisms and mammalian cells by using an ESR oximetry technique.     

Evidence for a nonoxidative mechanism of human natural killer (NK) cell cytotoxicity by using mononuclear effector cells from healthy donors and from patients with chronic granulomatous disease

In vitro natural killer (NK) activity expressed by blood mononuclear cells from patients with chronic granulomatous disease of childhood (CGD) was equivalent to that expressed by cells from normal, healthy volunteers. Because neutrophils and monocytes from these same donors exhibited extremely depressed oxidative functions, our data could be interpreted to show that a) NK cells derived from a unique and separate cellular lineage unaffected by the disease-related oxidative defect, or b) the in vitro cytolytic mechanism(s) of NK cells were not dependent on oxygen metabolites. These hypotheses were examined by using as NK effector cells large granular lymphocytes (LGL) from healthy donors whose monocytes and neutrophils had normal oxidative functions. Such functions were measured in the nitroblue tetrazolium dye reduction assay, which is a qualitative measurement of superoxide anion production; by reduction of ferric cytochrome c, a more specific and quantitative measurement of superoxide anion production; and in the luminol-enhanced chemiluminescence assay, an extremely sensitive measure of several reactive oxygen radicals, including superoxide anion, hydroxyl radical, and singlet oxygen. Whereas monocytes and neutrophils from healthy donors were readily stimulated with zymosan or phorbol myristate acetate (PMA) in each of these assays. LGL produced no detectable amounts of oxygen metabolites when co-incubated either with K562 erythroleukemia cells, PMA, E. coli endotoxin, or the calcium ionophore A23187. Thus, because NK cell activity is normal in CGD patients with major oxidative defects, and because no reactive oxygen metabolites could be detected in LGL that simultaneously exhibited potent NK activity, we conclude that in vitro NK activity by human mononuclear cells involves a lytic mechanism(s) independent of oxygen metabolites.     

Improvement of a microbial fuel cell performance as a BOD sensor using respiratory inhibitors

Studies were made to improve the performance of a microbial fuel cell (MFC) as a biochemical oxygen demand (BOD) sensor. The signal from MFCs decreased in the presence of electron acceptors of higher redox potential such as nitrate and oxygen. The addition of azide and cyanide did not change the signal in the absence of the electron acceptors. The respiratory inhibitors eliminated the inhibitory effects of the electron acceptors on the current generation from MFCs. Similar results were obtained using oligotrophic MFCs fed with an environmental sample that contained nitrate. The use of the respiratory inhibitors is therefore recommended for the accurate BOD measurement of environmental samples containing nitrate and/or oxygen with an MFC-type BOD sensor.     

The role of a bifunctional catalase-peroxidase KatA in protection of Agrobacterium tumefaciens from menadione toxicity

Agrobacterium tumefaciens is an aerobic plant pathogenic bacterium that is exposed to reactive oxygen species produced either as by-products of aerobic metabolism or by the defense systems of host plants. The physiological function of the bifunctional catalase-peroxidase (KatA) in the protection of A. tumefaciens from reactive oxygen species other than H(2)O(2) was evaluated in the katA mutant (PB102). Unexpectedly, PB102 was highly sensitive to the superoxide generator menadione. The expression of katA from a plasmid vector complemented the menadione-hypersensitive phenotype. A. tumefaciens possesses an additional catalase gene, a monofunctional catalase encoded by catE. Neither inactivation nor high-level expression of the catE gene altered the menadione resistance level. Moreover, heterologous expression of the catalase-peroxidase-encoding gene katG from Burkholderia pseudomallei, but not the monofunctional catalase gene katE from Xanthomonas campestris could restore normal levels of menadione resistance to PB102. A recent observation suggests that the menadione resistance phenotype involves increased activities of organic peroxide-metabolizing enzymes. Heterologous expression of X. campestris alkyl hydroperoxide reductase from a plasmid vector failed to complement the menadione-sensitive phenotype of PB102. The level of menadione resistance shows a direct correlation with the level of peroxidase activity of KatA. This is a novel role for KatA and suggests that resistance to menadione toxicity is mediated by a new, and as yet unknown, mechanism in A. tumefaciens.     

Reactive oxygen species may contribute to reduced endothelium-dependent dilation in rats fed high salt

In normotensive rats, an increase in dietary salt leads to decreased arteriolar responsiveness to acetylcholine (ACh) because of suppressed local nitric oxide (NO) activity. We evaluated the possibility that generation of reactive oxygen species in the arteriolar wall is responsible for this loss of NO activity. Arteriolar responses to iontophoretically applied ACh were examined in the superfused spinotrapezius muscle of Sprague-Dawley rats fed a low-salt (LS; 0.45%) or high-salt diet (HS; 7%) for 4-5 wk. Responses to ACh were significantly depressed in HS rats but returned to normal in the presence of the oxidant scavengers superoxide dismutase + catalase or 2,2,6, 6-tetamethylpiperidine-N-oxyl (TEMPO) + catalase. Arteriolar responses to the NO donor sodium nitroprusside were similar in HS and LS rats. Arteriolar and venular wall oxidant activity, as determined by reduction of tetranitroblue tetrazolium, was significantly greater in HS rats than in LS rats. Exposure to TEMPO + catalase reduced microvascular oxidant levels to normal in HS rats. These data suggest that a high-salt diet leads to increased generation of reactive oxygen species in striated muscle microvessels, and this increased oxidative state may be responsible for decreased endothelium-dependent responses associated with high salt intake.     

Catalase, glutathione peroxidase, and superoxide dismutase in the rete mirabile and gas gland epithelium of six species of marine fishes

Rete mirabile and gas gland epithelium from the swim bladders of six species of marine fishes were assayed for catalase, glutathione peroxidase, and superoxide dismutase activity. Correlation of the results of these assays with measurements of the concentration of oxygen in the lumen of the normal steady state swim bladders revealed that swim bladders in species containing higher levels of oxygen also exhibited higher levels of superoxide dismutase activity in the rete mirabile/gas gland epithelium region. There appeared to be no correlation between oxygen concentration and the level of catalase or glutathione peroxidase activity. Induction of the inflatory reflex in Opsanus tau by a single deflation of the swim bladder resulted in an increase in the percent of oxygen in the swim bladder lumen 18 to 24 hours later, but this was not accompanied by any significant increases in antioxidant enzyme activity. Swim bladders that were deflated three times at 24-hour intervals showed further increases in oxygen concentration at the end of the 72-hour period but no alteration in superoxide dismutase activity.     

Superoxide dismutase and catalase in Azotobacter vinelandii grown in continuous culture at different dissolved oxygen concentrations

Superoxide dismutase and catalase activities were studied in Azotobacter vinelandii grown diazotrophically at different ambient oxygen concentrations in continuous culture. Activities were expressed either as specific activity or activity per cell. Specific superoxide dismutase activity increased by a factor of 1.6 with increasing oxygen concentration from about 1% to 90% air saturation of the growth medium whereas specific catalase activity increased only slightly, if at all. Since cell volumes increased in parallel to increases in the oxygen concentration cellular superoxide dismutase activities increased by a factor of 4.3 while cellular catalase activities increased by a factor of 3.3. Under all conditions only the Fe-containing form of superoxide dismutase was detected. The possible function of these enzymes in the protection nitrogenase from oxygen damage is discussed.Abbreviation SOD superoxide dismutase     

Catalase activity and the survival of Pseudomonas putida, a root colonizer, upon treatment with peracetic acid

Peracetic acid is used as a sterilant in several industrial settings. Cells of a plant-colonizing bacterium, Pseudomonas putida in liquid suspension, were more sensitive to killing by peracetic acid when they lacked a major catalase activity, catalase A. Low doses of peracetic acid induced promoter activity of the gene encoding catalase A and increased total catalase specific activity in cell extracts. Microbes present in native agricultural soils rapidly degraded the active oxygen species present in peracetic acid. The simultaneous release of oxygen was consistent with a role for catalase in degrading the hydrogen peroxide that is part of the peracetic acid-equilibrium mixture. Amendment of sterilized soils with wild-type P. putida restored the rate of degradation of peracetic acid to a higher level than was observed in the soils amended with the catalase A-deficient mutant. The association of the bacteria with the plant roots resulted in protection of the wild-type as well as the catalase-deficient mutant from killing by peracetic acid. No differential recovery of the wild-type and catalase A mutant of P. putida was observed from roots after the growth matrix containing the plants was flushed with peracetic acid.     

Catalase, superoxide dismutase, and the production of O2-sensitive mutants of Bacillus coagulans

A number of facultatively anaerobic members of the genus Bacillus were screened for their catalase, diaminobenzidine peroxidase, and superoxide dismutase activities. A strain of Bacillus coagulans (7050) lacking peroxidatic activity and containing single catalatic and superoxide dismutase activities was selected. Responses of the superoxide dismutase activity and catalase level to the partial pressure of oxygen, and Fe and Mn levels, as well as to aerobic and fermentative metabolism, were determined. There appeared to be a relationship between high endogenous catalase levels and the high H2O2 evolution and KCN insensitivity of B. coagulans respiration. Bacillus coagulans 7050 was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and screened for the expression of oxygen intolerance. All of the 38 stable oxygen sensitive mutants obtained had very low or completely absent catalatic activity and catalase protein. No mutant lacked superoxide dismutase, although five showed significantly lowered levels of the enzyme. Exogenous bovine liver catalase restored aerotolerance and reduced cell pleomorphism in the mutants.     

Oxygen-dependent inactivation of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts of Rhodospirillum rubrum and establishment of a model inactivation system with purified enzyme.

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/O) was inactivated in crude extracts of Rhodospirillum rubrum under atmospheric levels of oxygen; no inactivation occurred under an atmosphere of argon. RuBP carboxylase activity did not decrease in dialyzed extracts, indicating that a dialyzable factor was required for inactivation. The inactivation was inhibited by catalase. Purified RuBPC/O is relatively oxygen stable, as no loss of activity was observed after 4 h under an oxygen atmosphere. The aerobic inactivation catalyzed by endogenous factors in crude extracts was mimicked by using a model system containing purified enzyme, ascorbate, and FeSO4 or FeCl3. Dithiothreitol was found to substitute for ascorbate in the model system. Preincubation of the purified enzyme with RuBP led to enhanced inactivation, whereas Mg2+ and HCO3- significantly protected against inactivation. Unlike the inactivation catalyzed by endogenous factors from extracts of R. rubrum, inactivation in the model system was not inhibited by catalase. It is proposed that ascorbate and iron, in the presence of oxygen, generate a reactive oxygen species which reacts with a residue at the activation site, rendering the enzyme inactive.     

Properties of erythrocyte catalase from homozygotes and heterozygotes for Swiss-type acatalasemia

The unstable catalase variant found in the blood of individuals homozygous for Swiss-type acatalasemia and the enzyme species present in heterozygous carriers of this rare defect have been further characterized. The mutant enzyme isolated from acatalasemic red cells is considerably more heat labile and differs in electrophoretic mobility from the normal enzyme. Catalase preparations obtained from heterozygotes consist of an apparently uniform enzyme species, probably representing a molecular hybrid, with properties intermediate to those of the normal and the variant enzyme. However, antigenic identity of catalase from all three sources is observed. Model experiments indicate that hybrid catalase molecules can be produced by recombining normal and variant dimer subunits. Fractionation of erythrocytes according to density and age shows that most of the residual catalase activity is localized in juvenile acatalasemic cells, whereas in normal and heterozygous individuals the catalase activity level does not alter significantly during the life span of the red cells. These findings agree with the observation that there is no gene dosage in heterozygotes, their catalase activity values falling within the normal range.     

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.     

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 mm 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 m was calculated.     

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.     

The peroxidatic and catalatic activity of catalase in normal and acatalasemic mouse liver

Monomeric, dimeric and tetrameric forms of mouse liver catalase have been shown to express peroxidatic activity while the tetrameric form expresses the catalic activity. Autosomally inherited acatalasemia, produced by X-ray irradiation of mice results in almost complete loss of catalic activity of catalase but has no effect on the peroxidatic activity. Liver catalase from normal and acatalasemic mice was purified by following the catalic and peroxidatic activity, respectively. Antiserum produced in rabbit against catalase from normal mouse completely precipitated the catalatic and peroxidatic activity from normal liver, and peroxidatic activity from the acatalasemic liver homogenate. Similar results were obtained when antiserum against peroxidase from acatalasemic mice was used. These studies indicate that acatalasemia in mice is due to a structural gene mutation which leads to synthesis of structurally altered catalase subunits. The altered subunits express peroxidatic activity but do not combine to form a tetramer which expresses catalatic activity.     

Characterization of the oxidase activity in mammalian catalase

Catalase is a highly conserved heme-containing antioxidant enzyme known for its ability to degrade hydrogen peroxide into water and oxygen. In low concentrations of hydrogen peroxide, the enzyme also exhibits peroxidase activity. We report that mammalian catalase also possesses oxidase activity. This activity, which is detected in purified catalases, cell lysates, and intact cells, requires oxygen and utilizes electron donor substrates in the absence of hydrogen peroxide or any added cofactors. Using purified bovine catalase and 10-acetyl-3,7-dihydroxyphenoxazine as the substrate, the oxidase activity was found to be temperature-dependent and displays a pH optimum of 7-9. The Km for the substrate is 2.4 x 10(-4) m, and Vmax is 4.7 x 10(-5) m/s. Endogenous substrates, including the tryptophan precursor indole, the neurotransmitter precursor beta-phenylethylamine, and a variety of peroxidase and laccase substrates, as well as carcinogenic benzidines, were found to be oxidized by catalase or to inhibit this activity. Several dietary plant micronutrients that inhibit carcinogenesis, including indole-3-carbinol, indole-3-carboxaldehyde, ferulic acid, vanillic acid, and epigallocatechin-3-gallate, were effective inhibitors of the activity of catalase oxidase. Difference spectroscopy revealed that catalase oxidase/substrate interactions involve the heme-iron; the resulting spectra show time-dependent decreases in the ferric heme of the enzyme with corresponding increases in the formation of an oxyferryl intermediate, potentially reflecting a compound II-like intermediate. These data suggest a mechanism of oxidase activity involving the formation of an oxygen-bound, substrate-facilitated reductive intermediate. Our results describe a novel function for catalase potentially important in metabolism of endogenous substrates and in the action of carcinogens and chemopreventative agents.