Catalase is regulated by ubiquitination and proteosomal degradation. Role of the c-Abl and Arg tyrosine kinases

Catalase is a major effector in the defense of aerobic cells against oxidative stress. Recent studies have shown that catalase activity is stimulated by the c-Abl and Arg tyrosine kinases. Little, however, is otherwise known about the mechanisms responsible for catalase regulation. The present work demonstrates that mouse cells deficient in both c-Abl and Arg exhibit increased catalase stability. The results also show that catalase is subject to ubiquitination and degradation by the 26S proteosome. Significantly, ubiquitination of catalase is dependent on c-Abl- and Arg-mediated phosphorylation of catalase on both Y231 and Y386. In concert with these results, human 293 cells expressing catalase mutated at Y231 and Y386 exhibit attenuated levels of reactive oxygen species when exposed to hydrogen peroxide. These findings indicate that, in addition to stimulating catalase activity, c-Abl and Arg promote catalase degradation in the oxidative stress response.     

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.     

Decrease of hepatic catalase level by treatment with diallyl sulfide and garlic homogenates in rats and mice

Diallyl sulfide (DAS) is a flavor compound derived from garlic and is active in the inhibition of chemically induced cytotoxicity and carcinogenicity in animal models. This study was conducted to examine the effects of the treatment of DAS and garlic homogenates on the activities of catalase, glutathione peroxidase, and superoxide dismutase. Male Sprague-Dawley rats were treated with DAS i.g. at daily doses of 50 or 200 mg/kg for 8 days, causing the hepatic catalase activity to decrease by 55 and 95%, respectively. Such a decrease in hepatic catalase activity was also observed when the DAS treatment was extended to 29 days. Western blot analysis showed that the DAS treatments resulted in corresponding decreases in the liver catalase protein level. No significant change in the catalase activity in the kidney, lung, and brain was observed with the treatments, but a slight decrease in heart catalase activity was observed. These treatments did not cause significant changes in superoxide dismutase and glutathione peroxidase activities in these tissues. Treatment with DAS at a daily dose of 200 mg/kg for 1-7 days resulted in a gradual decrease in the liver catalase activity to 5% of the control level, but it did not decrease the erythrocyte catalase activity. Treatment of rats with fresh garlic homogenates (2 or 4 g/kg, i.g., daily for 7 days) caused a 35% decrease in liver catalase activity. A/J mice treated with DAS and garlic homogenates also showed a decrease in the liver catalase activity. Diallyl sulfone (DASO2), a DAS metabolite, however, did not effectively decrease catalase activity in mice. The catalase activity was not inhibited by either DAS or DASO2 in vitro. The present results demonstrate that treatment with DAS and garlic homogenates decrease the hepatic catalase level in rats and mice.     

Immunotitration of the catalase in the blood of Japanese subjects and mice suffering from acatalasemia and hypocatalasemia.

Catalase in hemolysates of normal, heterozygous hypocatalasemic and acatalasemic Japanese was immunotitrated with an anti-human blood catalase rabbit serum. Equivalence points were calculated from the regression lines between catalase activity added and catalase activity remaining in the supernatant. Catalase activities at the equivalence points of Japanese normal, hypocatalasemia and acatalasemia were similar. The results indicate that the specific activities of catalase in the normal and of the variant bloods are identical. Catalase in hemolysates of normal and variant mice was immunotitrated with an anti-mouse liver catalase rabbit serum. In contrast to Japanese acatalasemic subject, the equivalence points of catalase in heterozygous hypocatalasemic, homozygous hypocatalasemic, acatalasemic and normal hemolysates were different, and the ratios of specific activity in these variant mice to that in normal were 0.72, 0.46 and 0.21, respectively. The differences in catalase activities at equivalence points were also supported by the statistical analysis on parameters of regression lines of catalase activities remaining in the supernatant on catalase activities added in the immunotitration. These findings suggest that the molecular properties of residual catalase of Japanese acatalasemia and those of mouse acatalasemia are entirely different.     

Dissociation of bovine and bacterial catalases by sodium n-dodecyl sulfate

The dissociation of beef liver and bacterial (Micrococcus lysodeikticus) catalases by the action of sodium n-dodecyl sulfate (SDS) has been investigated as a function of SDS concetration and time by ultracentrifugation. The rate of dissociation of beef liver catalase is found to be much faster than that for bacterial catalase in 25 mM SDS at pH 7.0. Beef liver catalase is dissociated into its four subunits after 24 h, whereas bacterial catalase is not completely dissociated after 36 days of incubation. The binding of SDS to beef liver catalase obeys a Hill equation with a cooperativity exponent of 2.0 and a binding constant of 440. The initial interaction of SDS with beef liver catalase can be detected by microcalorimetry, whereas the mixing of SDS with bacterial catalase is athermal. Bacterial catalase retains enzymic activity in the presence of SDS, whereas beef liver catalase is completely deactivated at SDS concentrations above 5 mM. Beef liver catalase is more sensitive to acid denaturation than bacterial catalase, and the rate of dissociation for both catalases is sixth-order in proton concentration. Comparison of the amino acid analysis of the two catalases shows that bacterial catalase has a smaller number of lysyl residues and a larger number of glutamyl residues than beef liver catalase. Taken together these structural differences could lead to a reduced affinity of bacterial catalase for the binding of SDS as observed.     

Study of the coinduction by fatty acids of catalase A and acyl-CoA oxidase in standard and mutant Saccharomyces cerevisiae strains

Evidence is presented that Saccharomyces cerevisiae can metabolize fatty acids via the inducible peroxisomal beta-oxidation pathway even when these acids are not the sole carbon source. The fatty acids of chain length of C10-C18 induce acyl-CoA oxidase simultaneously with catalase A but have no effect on catalase T and acyl-CoA dehydrogenase. The coinduction of both acyl-CoA oxidase and catalase A is recorded in strains with both active catalase A and T or displaying only catalase A activity. In mutants lacking catalase A, the induction of acyl-CoA oxidase is observed without a concomitant increase in catalase activity. After centrifugation in a linear Ficoll gradient of the particulate fraction from the cells grown on ethanol and oleate the activity of acyl-CoA oxidase cosediments with catalase A. The relationship of catalase A to acyl-CoA oxidase is discussed.     

Purification of glyoxysomal catalase and immunochemical comparison of glyoxysomal and leaf peroxisomal catalase in germinating pumpkin cotyledons

As a step to study the mechanism of the microbody transition (glyoxysomes to leaf peroxisomes) in pumpkin (Cucurbita sp. Amakuri Nankin) cotyledons, catalase was purified from glyoxysomes. The molecular weight of the purified catalase was determined to be 230,000 to 250,000 daltons. The enzyme was judged to consist of four identical pieces of the monomeric subunit with molecular weight of 55,000 daltons. Absorption spectrum of the catalase molecule gave two major peaks at 280 and 405 nanometers, showing that the pumpkin enzyme contains heme. The ratio of absorption at 405 and 280 nanometers was 1.0, the value being lower than that obtained for catalase from other plant sources. These results indicate that the pumpkin glyoxysomal catalase contains the higher content of heme in comparison with other plant catalase.

The immunochemical resemblance between glyoxysomal and leaf peroxisomal catalase was examined by using the antiserum specific against the purified enzyme preparation from pumpkin glyoxysomes. Ouchterlony double diffusion and immunoelectrophoretic analysis demonstrated that catalase from both types of microbodies cross-reacted completely whereas the immunotitration analysis showed that the specific activity of the glyoxysomal catalase was 2.5-fold higher than that of leaf peroxisomal catalase. Single radial immunodiffusion analysis showed that the specific activity of catalase decreased during the greening of pumpkin cotyledons.

     

Quantitative indices of the activity and the electrophoretic characteristics of staphylococcal catalase]

The average levels of activity of intracellular and extracellular catalase were determined. The activity of intracellular catalase was shown to be significantly higher than that of extracellular catalase, the average level of activity of extracellular catalase being higher in S. aureus than in S. epidermidis. In most of the strains one zone of extracellular catalase and two molecular forms of intracellular catalase were revealed by means of polyacrylamide gel electrophoresis.     

Purification and properties of recombinant rat catalase produced in Escherichia coli

Catalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.     

Effect of aminotriazole on the activity of catalase and glucose-6-phosphate dehydrogenase in tissues of two frog species--Rana ridibunda and Rana esculenta

Changes of the activity of catalase and glucose-6-phosphate dehydrogenase (G6PDH) during 48 hrs after intraperitoneal injection of 1.0, 0.5 and 0.1 mg aminotriazole per gram of body weight of two frog species as well as catalase inhibition by aminotriazole in vitro were investigated. Both aminotriazole concentration and species affiliation affected the catalase inhibition. The sensitivity of catalase from different tissues was decreased in the order: liver--kidney--lung--muscle--brain. The constant of half inhibition of lung catalase was significantly lower than that of liver and kidney catalase. The activity of G6PDH of AMT-treated frogs R. esculenta was higher comparing to control group. Possible ways of compensation of antioxidant defense under catalase inhibition are discussed.     

Correlation between the catalase level in tumor cells and their sensitivity to N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD)

N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD) exhibits selective cytotoxicity toward certain human tumor cell lines. 5-S-GAD has been shown to release hydrogen peroxide autonomously. Hydrogen peroxide is converted to water and oxygen by catalase. The purpose of this study is to determine whether or not 5-S-GAD exhibits selective cytotoxicity toward tumor cells with low catalase levels, but not toward ones with high catalase levels. We transfected MDA-MB-435S cells, which are sensitive to 5-S-GAD, with catalase cDNA to establish high catalase producer cells, and then examined their 5-S-GAD sensitivity. Similarly, we repressed catalase expression in T47D cells, which are insensitive to 5-S-GAD, by catalase RNA interference to create low catalase producer cells, and then examined their 5-S-GAD sensitivity. We show that the overexpression of catalase made MDA-MB-435S cells insensitive to 5-S-GAD, whereas the suppression of catalase made T47D cells sensitive to 5-S-GAD. The cellular catalase level was found to be crucial for cell sensitivity to 5-S-GAD.     

Activity and hematin content of catalase from greening sunflower cotyledons

The specific activity of catalase purified from the peroxisomes of sunflower cotyledons declines in parallel with the total cotyledonary catalase activity during the transition of peroxisomes from glyoxysomal to leaf peroxisomal function. The hematin content of the purified catalase however, remains constant at 4 hematin groups per catalase molecule. The absorbance coefficients of catalase at 404 and 280 nm were determined to be 372 and 540/mM/cm, respectively.     

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.     

Immobilization and kinetics of catalase onto magnesium silicate

Bovine liver catalase was immobilized covalently with glutaraldehyde, or glutaraldehyde+3-aminopropionic acid as a spacer, onto magnesium silicate. The coupling time was determined as 2 h for immobilization. The pH and temperature optima as well as the changes in the kinetics (K_m, V_max, E_a) of the immobilized catalase was observed and discussed. Immobilized catalase preparations showed higher storage stabilities than free catalase. The half-life of free catalase, catalase immobilized via glutaraldehyde and catalase immobilized via glutaraldehyde+spacer were calculated as 2, 55 and 10 days at room temperature and 4, 85 and 107 days at 5 °C, respectively. The operational stability of the catalase immobilized via glutaraldehyde was higher than the catalase immobilized via glutaraldehyde+spacer. The remaining activity of the catalase immobilized via glutaraldehyde was about 90% and that of the catalase immobilized via glutaraldeyde+spacer was about 30% after 20 cycles of batch operation.     

Catalase Synthesis and Turnover during Peroxisome Transition in the Cotyledons of Helianthus annuus L

Based on measurements of total catalase hematin and the degradation constants of catalase hematin, zero order rate constants for the synthesis of catalase were determined during the development of sunflower cotyledons (Helianthus annuus L.). Catalase synthesis reached a sharp maximum of about 400 picomoles hematin per day per cotyledon at day 1.5 during the elaboration of glyoxysomes in the dark. During the transition of glyoxysomes to leaf peroxisomes (greening cotyledons, day 2.5 to 5) catalase synthesis was constant at a level of about 30 to 40 picomoles hematin per day per cotyledon. In the cotyledons of seedlings kept in the dark (day 2.5 to 5) catalase synthesis did not exceed 10 picomoles hematin per day per cotyledon. During the peroxisome transition in the light, total catalase hematin was maintained at a high level, whereas total catalase activity rapidly decreased. In continuous darkness, total catalase hematin decreased considerably from a peak at day 2. The results show that both catalase synthesis and catalase degradation are regulated by light. The turnover characteristics of catalase are in accordance with the concept that glyoxysomes are transformed to leaf peroxisomes as described by the one population model and contradict the two population model and the enzyme synthesis changeover model which both postulate de novo formation of the leaf peroxisome population and degradation of the glyoxysome population.     

Mitochondrial catalase and oxidative injury

Mitochondria dysfunction induced by reactive oxygen species (ROS) is related to many human diseases and aging. In physiological conditions, the mitochondrial respiratory chain is the major source of ROS. ROS could be reduced by intracellular antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase as well as some antioxidant molecules like glutathione and vitamin E. However, in pathological conditions, these antioxidants are often unable to deal with the large amount of ROS produced. This inefficiency of antioxidants is even more serious in mitochondria, because mitochondria in most cells lack catalase. Therefore, the excessive production of hydrogen peroxide in mitochondria will damage lipid, proteins and mDNA, which can then cause cells to die of necrosis or apoptosis. In order to study the important role of mitochondrial catalase in protecting cells from oxidative injury, a HepG2 cell line overexpressing catalase in mitochondria was developed by stable transfection of a plasmid containing catalase cDNA linked with a mitochondria leader sequence which would encode a signal peptide to lead catalase into the mitochondria. Mitochondria catalase was shown to protect cells from oxidative injury induced by hydrogen peroxide and antimycin A. However, it increased the sensitivity of cells to tumor necrosis factor-alpha-induced apoptosis by changing the redox-oxidative status in the mitochondria. Therefore, the antioxidative effectiveness of catalase when expressed in the mitochondrial compartment is dependent upon the oxidant and the locus of ROS production.     

On the synthesis and degradation of the multiple forms of catalase in mouse liver: effects of aminotriazole and p-chlorophenoxyisobutric acid ethyl ester

Aspects of the synthesis and degradation of the multiple forms of catalase in mouse liver have been investigated. The kinetics of return of catalase after aminotriazole inhibition indicated a product-precursor relationship between the granular and supernatant pools of the enzyme, and electrophoretic resolution of the individual heteromorphs of catalase during this treatment served to substantiate this relationship and indicated, in addition, that aminotriazole-inhibited catalase may be partially reactivated in the cytosol. Changes in the activity of mouse liver and kidney catalases after the administration of chlorophenoxyisobutyric acid ethyl ester (CPIB) were also monitored. After an initial decline in activity, a rapid increase to an elevated steady state occurred, with an approximately threefold increase in the liver and twofold increase in the kidney. Subcellular fractionation of the livers of CPIB-treated mice showed a massive initial increase in the supernatant pool of catalase, accompanied by a steady decrease in the activity of the peroxisomal pools. Activity increased in the peroxisomal pools at later stages of treatment, but even after a new CPIB-induced steady state was achieved, the supernatant pool of catalase remained grossly elevated. Electrophoresis of the individual heteromorphs of catalase in the supernatant after CPIB treatment showed a predominance of the most cathodal migrating forms, and turnover studies demonstrated that catalase in CPIB-treated animals exhibited a substantially lowered rate of degradation by comparison with normal animals. These results have been discussed in relation to the intracellular sequestration and turnover characteristics of catalase.