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.     

Is the cytosolic catalase induced by peroxisome proliferators in mouse liver on its way to the peroxisomes?

Dietary treatment of male C57B1/6 mice with clofibrate, nafenopin or WY-14.643 resulted in a modest (at most 2-fold) increase in the total catalase activity in the whole homogenate and mitochondrial fraction prepared from the livers of these animals. On the other hand, the catalase activity recovered in the cytosolic fraction was increased 12- to 18-fold, i.e. 30-35% of the total catalase activity in the hepatic homogenate was present in the high-speed supernatant fraction after treatment with these peroxisome proliferators. A study of the time course of the changes in peroxisomal and cytosolic catalase activities demonstrated that the peroxisomal activity both increased upon initiation of exposure and decreased after termination of treatment several days after the increase and decrease, respectively, in the corresponding cytosolic activity. This finding suggests that the cytosolic catalase may be on its way to incorporation into peroxisomes.     

Enhanced heme oxygenase activity increases the antioxidant defense capacity of guinea pig liver upon acute cobalt chloride loading: comparison with rat liver

Changes in the activity of so-called oxidative stress defensive enzymes, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and heme oxygenase, as well as changes in lipid peroxidation and reduced glutathione levels, were measured in guinea pig and rat liver after acute cobalt loading. Cobalt chloride administration produced a much higher degree of lipid peroxidation in guinea pig than in rat liver compared with the control animals. The intrahepatic reduced glutathione content in control guinea pig was higher than that in rat, but was equally decreased in both species after cobalt administration. The enzymatic scavengers of free radicals, superoxide dismutase, catalase and glutathione peroxidase, were significantly decreased in rat liver after acute cobalt loading, and as a compensatory reaction, the heme oxygenase activity was increased (seven-fold). In guinea pig liver, only superoxide dismutase activity was depleted in response to cobalt-induced oxidative stress, while catalase and glutathione peroxidase were highly activated and the heme oxygenase activity was dramatically increased (13-fold). It is assumed that enhanced heme oxygenase activity may have important antioxidant significance by increasing the liver oxidative-stress defense capacity.     

Catalase induction protects Haemonchus contortus against hydrogen peroxide in vitro

The response of Haemonchus contortus to oxidative stress in vitro was examined by measuring catalase activities in adult and L4 stage worms exposed to hydrogen peroxide generated by a glucose/glucose oxidase system. Adult nematodes showed increases of up to 2.3-fold in catalase activity after 42 h exposure to the peroxide. L4 nematodes showed up to 4.6-fold induction. A two-stage dose-response was apparent, with catalase activities increasing as the peroxide levels increased, before a return to control levels at higher peroxide concentrations, most likely reflecting a balance between induction and toxicity of the inducing agent itself. Adult nematodes exposed to low levels of peroxide for 24h (hence, having enhanced catalase activities) showed an ability to tolerate subsequent exposure to toxic levels of the peroxide compared to worms with no pre-exposure. An increase of up to approximately threefold in the LC(50) of the hydrogen peroxide generating system was observed after hydrogen peroxide pre-exposure. This indicates that exposure to low oxidant levels lead to an increase in defensive enzyme activities, which allows the nematode to survive subsequent oxidant threats more effectively. The ability of H contortus to increase its catalase activity may be crucial in allowing it to respond to the production of reactive oxygen species by host phagocytes in vivo.     

Correlation between melanogenic and catalase activity in in vitro human melanocytes: a synergic strategy against oxidative stress

UV-induced DNA damage can lead to melanoma, the most dangerous form of skin cancer. Understanding the mechanisms employed by melanocytes to protect against UV is therefore a key issue. In melanocytes, catalase is the main enzyme responsible for degrading hydrogen peroxide and we have previously shown that that low basal levels of catalase activity are associated with the light phototype in in vitro and ex vivo models. Here we investigate the possible correlation between its activity and melanogenesis in primary cultures of human melanocytes. We show that while the total melanin concentration is directly correlated to the level of pigmentation, the more the degree of pigmentation increased, the lower the proportion of pheomelanin present. Moreover, in human melanocytes in vitro, catalase-specific mRNA, protein and enzymatic activity were all directly correlated with total cellular melanin content. We also observed that immediately after a peroxidative treatment, the increase in reactive oxygen species was inversely associated with pigmentation level. Darkly pigmented melanocytes therefore possess two protective strategies represented by melanins and catalase activity that are likely to act synergistically to counteract the deleterious effects of UV radiation. By contrast, lightly pigmented melanocytes possess lower levels of melanogenic and catalase activity and are therefore more susceptible to accumulate damage after UV exposition.     

Peroxisomes of the rat cardiac and soleus muscles increase after starvation. A biochemical and immunocytochemical study

We have investigated the change of catalase activity in the homogenates of rat cardiac and skeletal muscles. After 7 days' starvation, the catalase activity of heart increased about 3-fold and that of soleus muscle enhanced 2-fold higher than that of control rats. Immunoblot analysis of catalase showed a single band in the homogenates of cardiac and soleus muscles and increase of catalase antigen after starvation. Light microscopic immunoenzyme staining showed that after starvation catalase positive granules markedly increased in both the cardiac and soleus muscle. Quantitative analysis of the staining showed that number of the granules per 100 microns 2 of tissue section was about 1.4-fold in the soleus muscle and 1.7-fold in the cardiac muscle after starvation. By electron microscopy of alkaline DAB staining, we confirmed that the granules were peroxisomes, which increased in both number and size. Furthermore, we stained the peroxisomes for catalase by a protein A-gold technique. Labeling density (gold particles/micron 2) of the cardiac and soleus muscles from the starved rat increased approximately 1.4 times as much as that of normal animal. When the numerical density is multiplied by the labeling density, the values are largely consistent with the enhancement of catalase activity. These results show that increase in the catalase activity of the muscle tissue after starvation is caused by increase in number and size of peroxisomes.     

On the relationship of coral allene oxide synthase to catalase. A single active site mutation that induces catalase activity in coral allene oxide synthase

A heme domain of coral allene oxide synthase (cAOS) catalyzes the formation of allene oxide from fatty acid hydroperoxide. Although cAOS has a similar heme active site to that of catalase, cAOS is completely lacking in catalase activity. A close look at the hydrogen-bonding possibilities around the distal His in cAOS suggested that the imidazole ring is rotated by 180 degrees relative to that of catalase because of the hydrogen bond between Thr-66 and the distal His-67. This could contribute to the functional differences between cAOS and catalase, and to examine this possibility, we mutated Thr-66 in cAOS to Val, the corresponding residue in catalase. In contrast to the complete absence of catalase activity in wild type (WT) cAOS, T66V had a modest catalase activity. On the other hand, the mutation suppressed the native enzymatic activity of the formation of allene oxide to 14% of that of WT cAOS. In the resonance Raman spectrum, whereas WT cAOS has only a 6-coordinate/high spin heme, T66V has a 5-coordinate/high spin heme as a minor species. Because catalase adopts a 5-coordinate/high spin structure, probably the 5-coordinate/high spin portion of T66V showed the catalase activity. Furthermore, in accord with the fact that the CN affinity of catalase is higher than that of WT cAOS, the CN affinity of T66V was 8-fold higher than that of WT cAOS, indicating that the mutation could mimic the heme active site in catalase. We, therefore, propose that the hydrogen bond between Thr-66 and distal His-67 could modulate the orientation of distal His, thereby regulating the enzymatic activity in cAOS.     

Alterations in Protective Enzymes Against Peroxidation in the Central and Peripheral Nervous System of Control and Dysmyelinating Mutant Mice

The activities of peroxide-detoxifying enzymes such as superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, and catalase were measured in the nervous system of neurological dysmyelinating mutants: quaking (Qk), shiverer (Shi), and trembler (Tr) mice. Cu/Zn-SOD activity was higher in the cerebellum of Qk and Shi mice (by 53% and 106%, respectively) in comparison with controls, but it was the same in the cerebellum of Tr mice and their corresponding controls. In contrast, there was no difference in the level of Cu/Zn-SOD activity in the cerebrum of Qk, Shi, and Tr mice and their respective controls. Mn-SOD activity was the same among all the mutants compared to control animals in both cerebrum and cerebellum. In Shi cerebellum, glutathione peroxidase and glutathione reductase activities were slightly decreased (a 21.6% and a 13.2% diminution, respectively), whereas catalase activity in cerebrum and cerebellum was the same among mutants and control mice. In the sciatic nerve from Tr mice, all the enzymatic activities were enhanced: sixfold increase for total SOD, and 2.4-fold, 3.5-fold, and 1.8-fold increase for glutathione peroxidase, glutathione reductase, and catalase, respectively.     

Peroxisomes of the rat cardiac and soleus muscles increase after starvation

Summary We have investigated the change of catalase activity in the homogenates of rat cardiac and skeletal muscles. After 7 days' starvation, the catalase activity of heart increased about 3-fold and that of soleus muscle enhanced 2-fold higher than that of control rats. Immunoblot analysis of catalase showed a single band in the homogenates of cardiac and soleus muscles and increase of catalase antigen after starvation. Light microscopic immunoenzyme staining showed that after starvation catalase positive granules markedly increased in both the cardiac and soleus muscle. Quantitative analysis of the staining showed that number of the granules per 100 m² of tissue section was about 1.4-fold in the soleus muscle and 1.7-fold in the cardiac muscle after starvation. By electron microscopy of alkaline DAB staining, we confirmed that the granules were peroxisomes, which increased in both number and size. Furthermore, we stained the peroxisomes for catalase by a protein A-gold technique. Labeling density (gold particles/m²) of the cardiac and soleus muscles from the starved rat increased approximately 1.4 times as much as that of normal animal. When the numerical density is multiplied by the labeling density, the values are largely consistent with the enhancement of catalase activity. These results show that increase in the catalase activity of the muscle tissue after starvation is caused by increase in number and size of peroxisomes.     

Oxidative stress response of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> to hydrogen peroxide,paraquat and pressure

The aim of this work was to study the oxidative stress response of Kluyveromyces marxianus to hydrogen peroxide (50 mM), paraquat (1 mM), an increase in air pressure (120 kPa, 600 kPa) and pure oxygen pressure (120-600 kPa) in a pressurized bioreactor. The effect of these oxidants on metabolism and on the induction of antioxidant enzymes was investigated. The exposure for 1 h of K. marxianus at exponential growth phase with either H(2)O(2) or paraquat, under air pressure of 120 kPa or 600 kPa, induced an increase in both superoxide dismutase (SOD) and glutathione reductase (GR) content. SOD induction by the chemical oxidants was independent of the air pressure values used. A 2-fold increase in SOD activity was observed after 1 h of exposure to H(2)O(2) and a 3-fold increase was obtained by the presence of paraquat, with both air pressures studied. In contrast, GR activity was raised 1.7-fold by the exposure to both chemicals with 120 kPa, but a 2.4-fold GR induction was obtained with 600 kPa. As opposed to Saccharomyces cerevisiae, catalase was not induced and was even lower than the normal basal levels. This antioxidant enzyme seemed to be inhibited under increasing oxygen partial pressure. The cells showed a significant increase in SOD and GR activity levels, 4.7-fold and 4.4-fold, when exposed for 24 h to 120 kPa pure oxygen pressure. This behaviour was even more patent with 400 kPa. However, whenever cells were previously exposed to low air pressures, low enzymatic activity levels were measured after subsequent exposure to pure oxygen pressure.     

以Fe(III) 改性胶原纤维为载体固定过氧化氢酶

将胶原纤维用三价铁改性后作为载体,通过戊二醛的交联作用将过氧化氢酶固定在该载体上。制备的固定化过氧化氢酶蛋白固载量为16.7 mg/g,酶活收率为35％。研究了固定化酶与自由酶的最适pH、最适温度、热稳定性、贮存稳定性及操作稳定性。结果表明：过氧化氢酶经此法固定化后,最适pH及最适温度与自由酶相同,分别为pH 7.0和25 ℃;但固定化酶的热稳定性显著提高,在75 ℃保存5 h后,仍能保留30％的活力,而自由酶则完全失活;固定化酶在室温下保存12 d后,酶活力仍保持在88％以上,而自由酶在此条件下则完全失     

Acquired tolerance to oxidative stress in Bifidobacterium longum 105-A via expression of a catalase gene

For improvement of tolerance to oxidative stress in Bifidobacterium longum 105-A, we introduced the Bacillus subtilis catalase gene (katE) into it. The transformant showed catalase activity (39 U/mg crude protein) in the intracellular fraction, which increased survival by ～100-fold after a 1-h exposure to 4.4 mM H(2)O(2), decreased de novo H(2)O(2) accumulation, and increased survival in aerated cultures by 10(5)-fold at 24 h. The protection level was better than that conferred by exogenously added catalase.     

Catalase transgenic mice: characterization and sensitivity to oxidative stress

The role of catalase in the antioxidant defense system was studied using transgenic mice [Tg(CAT)] harboring a human genomic clone containing the entire human CAT gene. Catalase activity was 2-fold higher in the tissues of hemizygous [Tg(CAT)(+/o)] mice and 3- to 4-fold higher in the tissues of homozygous [Tg(CAT)(+/+)] mice compared to wild type mice. The human CAT transgene was expressed in a tissue-specific pattern that was similar to the endogenous catalase gene. The levels of other major antioxidant enzymes were not altered in the tissues of the transgenic mice. Hepatocytes and fibroblasts from the Tg(CAT)(+/+) mice were more resistant to hydrogen peroxide-induced cell death but were more sensitive to paraquat and TNFalpha toxicity. Fibroblasts from the Tg(CAT)(+/+) mice showed reduced growth rate in culture without treatment and reduced colony-forming capability after gamma-irradiation compared to fibroblasts from wild type mice. In addition, the Tg(CAT)(+/+) animals were more sensitive to gamma-irradiation.     

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.     

Weak antioxidant defenses make the heart a target for damage in copper-deficient rats

Copper deficiency causes more salient pathologic changes in the heart than in the liver of rats. Although oxidative stress has been implicated in copper deficiency-induced pathogenesis, little is known about the selective toxicity to the heart. Therefore, we examined the relationship between the severity of copper deficiency-induced oxidative damage and the capacity of antioxidant defense in heart and liver to investigate a possible mechanism for the selective cardiotoxicity. Weanling rats were fed a purified diet deficient in copper (0.4 μg/g diet) or one containing adequate copper (6.0 μg/g diet) for 4 weeks. Copper deficiency induced a 2-fold increase in lipid peroxidation in the heart (thiobarbituric assay) but did not alter peroxidation in the liver. The antioxidant enzymatic activities of superoxide dismutase, catalase, and glutathione peroxidase were, respectively, 3-, 50- and 1.5-fold lower in the heart than in the liver, although these enzymatic activities were depressed in both organs by copper deficiency. In addition, the activity of glutathione reductase was 4 times lower in the heart than in the liver. The data suggest that a weak antioxidant defense system in the heart is responsible for the relatively high degree of oxidative damage in copper-deficient hearts.     

The oxidation of chiral alcohols catalyzed by catalase in organic solvents

The catalytic properties of bovine liver catalase have been investigated in organic solvents. In tetrahydrofuran, dioxane, and acetone (all containing 1% to 3% of water), the enzyme breaks down tert-butyl hydroperoxide several fold faster than in pure water. Furthermore, the rate of catalase-catalyzed production of tert-butanol from tert-butyl hydroperoxide increases more than 400-fold upon transition from aqueous buffer to ethanol as the reaction medium. The mechanistic rationale for this striking effect is that in aqueous buffer the rate-limiting step of the enzymatic process involves the reduction of catalase's compound I by tert-butyl hydroperoxide. In ethanol, and additional step in the reaction scheme becomes available in which ethanol, greatly outcompeting the hydroperoxide, is oxidized by compound I regenerating the free enzyme. In solvents, such as acetonitrile or tetrahydrofuran, which themselves are not oxidizable by compound I, catalase catalyzes the oxidation of numerous primary and secondary alcohols with tert-butyl hydroperoxide to the corresponding aldehydes or ketones. The enzymatic oxidation of some chiral alcohols (2,3-butanediol, citronellol, and menthol) under these conditions occurs enantioselectively. Examination of the enantioselectivity for the oxidation of 2,3-butanediol in a series of organic solvents reveals a considerable solvent dependence. (c) 1995 John Wiley & Sons, Inc.     

Free-radical formation by mitomycin C and its novel analogs in cardiac microsomes and the perfused rat heart

Using a spin-trapping technique, we have examined free-radical formation by mitomycin C and its analogs, BMY 25282 and BMY 25067, in rat cardiac microsomes and isolated perfused rat hearts. All three drugs stimulated 2--4-fold OH radical formation in cardiac microsomes which was inhibited by SOD and catalase. Superoxide anion radical was also detected in the presence of diethylenetetraaminopentaacetic acid. Addition of DMSO yielded methyl radicals, thus indicating the production of free OH under these conditions. Similar stimulation of OH formation (2--3-fold) in the perfusates from rat hearts was detected with all three drugs. Perfusion with catalase (550 U/ml) completely suppressed the OH signal both in the presence and absence of the drugs, thus suggesting the intermediacy of hydrogen peroxide. However, BMY 25067-induced OH formation was more sensitive to inhibition by superoxide dismutase (SOD) and the iron chelator ICRF-187. Perfusion with DMSO produced methyl radicals at the expense of OH in the presence of all three drugs. SOD and catalase inhibited DMPO-OH signals, indicating that most of the OH formation was extracellular in this setting. While mitomycin C and BMY 25067 (up to 10 microM) did not affect the heart rate, perfusion with 10 microM BMY 25282 caused acute arrhythmia and cardiac standstill within 20 min. An initial surge in OH formation (2-fold) accompanied this cardiotoxic effect. Both the arrhythmia and the free radical signal were partially blocked by SOD, catalase and ICRF-187, indicating that iron-dependent oxygen radical formation from BMY-25282 (and possibly other compounds) is involved, in part, in inducing toxic manifestations in the rat heart and possibly in clinic.     

Enzymatic glutathione production using metabolically engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> as a whole-cell biocatalyst

We developed a novel enzymatic glutathione (GSH) production system using Saccharomyces cerevisiae as a whole-cell biocatalyst, and improved its GSH productivity by metabolic engineering. We demonstrated that the metabolic engineering of GSH pathway and ATP regeneration can significantly improve GSH productivity by up to 1.7-fold higher compared with the parental strain, respectively. Furthermore, the combination of both improvements in GSH pathway and ATP regeneration is more effective (2.6-fold) than either improvement individually for GSH enzymatic production using yeast. The improved whole-cell biocatalyst indicates its great potential for applications to other kinds of ATP-dependent bioproduction.     

Relationship Between Catalase and Life Span in Recombinant Inbred Strains of Caenorhabditis elegans

Johnson and Wood constructed recombinant inbred strains of Caenorhabditis elegans with life spans ranging from 10 to 31 days. Using these strains, we have demonstrated previously that hyperoxia and methyl viologen inhibited development at rates inversely correlated with life span. The growth rates of the short-lived recombinant inbred strains were more profoundly inhibited by oxidative stress than were those of the long-lived strains. Here we report a positive correlation between life span and catalase levels in these same strains. Specifically, when compared to short-lived strains at 10 days after fertilization, the long-lived strains possessed higher levels of total enzymatic catalase. Northern blots indicated a similar relationship between life span and clt-1mRNA (the cytosolic catalase). This suggests that at least some of the polygenes that influence life span are also responsible for regulating gene expression of catalase, an important defense component against oxidative stress.     

Role of mitochondrial electron transport chain complexes in capsaicin mediated oxidative stress leading to apoptosis in pancreatic cancer cells

We evaluated the mechanism of capsaicin-mediated ROS generation in pancreatic cancer cells. The generation of ROS was about 4-6 fold more as compared to control and as early as 1 h after capsaicin treatment in BxPC-3 and AsPC-1 cells but not in normal HPDE-6 cells. The generation of ROS was inhibited by catalase and EUK-134. To delineate the mechanism of ROS generation, enzymatic activities of mitochondrial complex-I and complex-III were determined in the pure mitochondria. Our results shows that capsaicin inhibits about 2.5-9% and 5-20% of complex-I activity and 8-75% of complex-III activity in BxPC-3 and AsPC-1 cells respectively, which was attenuable by SOD, catalase and EUK-134. On the other hand, capsaicin treatment failed to inhibit complex-I or complex-III activities in normal HPDE-6 cells. The ATP levels were drastically suppressed by capsaicin treatment in both BxPC-3 and AsPC-1 cells and attenuated by catalase or EUK-134. Oxidation of mitochondria-specific cardiolipin was substantially higher in capsaicin treated cells. BxPC-3 derived ρ(0) cells, which lack mitochondrial DNA, were completely resistant to capsaicin mediated ROS generation and apoptosis. Our results reveal that the release of cytochrome c and cleavage of both caspase-9 and caspase-3 due to disruption of mitochondrial membrane potential were significantly blocked by catalase and EUK-134 in BxPC-3 cells. Our results further demonstrate that capsaicin treatment not only inhibit the enzymatic activity and expression of SOD, catalase and glutathione peroxidase but also reduce glutathione level. Over-expression of catalase by transient transfection protected the cells from capsaicin-mediated ROS generation and apoptosis. Furthermore, tumors from mice orally fed with 2.5 mg/kg capsaicin show decreased SOD activity and an increase in GSSG/GSH levels as compared to controls. Taken together, our results suggest the involvement of mitochondrial complex-I and III in capsaicin-mediated ROS generation and decrease in antioxidant levels resulting in severe mitochondrial damage leading to apoptosis in pancreatic cancer cells.