Cytochemical study of catalase activity in Candida boidinii during incubation on methanol

Catalase activity of the methanol-assimilating yeast Candida boidinii M-363 was determined cytochemically and biochemically. Electron microscopic investigations on ultrathin sections were made on cells from 16, 24, and 48h batch cultures in nutrient medium with methanol (or glucose as a control) as the sole source of carbon and energy. The electron-dense oxidation product of 3,3′-diaminobenzidine was found predominantly in the mitochondrial cristae and membranes. The mitochondria were increased in number, enlarged, sometimes aggregated, with variable form and size and they characteristically developed when the strain was grown on methanol. The significant development of these organelles and their intensive DAB staining correlated with the considerable increase in catalase activity. Biochemically, catalase in the cell-free extract was determined to be maximal along the exponential growth phase of the strain during its incubation on methanol. Enzyme analysis of the heavy mitochondrial fraction showed that it possessed catalase activity but not peroxidase activity. The results showed that not only peroxisomes but also mitochondria may be structurally and functionally responsible for the high catalase activity of some methanol-assimilating yeasts. What is more, the contribution of the mitochondria to the utilization of methanol may be significant.     

Efficient Expression of Peroxidatic Activity of Catalase in the Coupled System with Glucose Oxidase—a model of Yeast Peroxisomes

Catalase functioned exclusively to degrade hydrogen peroxide in a reaction mixture containing methanol and hydrogen peroxide, while, when the enzyme was coupled with glucose oxidase, successful conversion of methanol to formaldehyde occurred at the optimized ratio of glucose oxidase to catalase: activity, 1.0 × 10 ^-3; number of molecules, 1.3; protein content, 1. These values in the coupled system were very similar to the ratio of alcohol oxidase to catalase in peroxisomes, one of the subcellular organelles from a methanol-assimilating yeast, Kloeckera sp. 2201, in which these enzymes were coupled to metabolize methanol efficiently. The presence of the optimum ratio in the coupled system in vitro was confirmed by the kinetic analysis of the expression of the peroxidatic activity of catalase coupled with glucose oxidase. Construction of the immobilized system of the coupled enzymes at the optimum ratio demonstrated that the oxidation of methanol through the peroxidatic function of catalase could be continuously and stably operated, the results indicating the usefulness of the system as a model of yeast peroxisomes. Thus, the coupled reaction with glucose oxidase brought out the latent function of catalase, which could not be expected in the system including only catalase.     

Alcohol oxidase and catalase in peroxisomes of methanol-grown Candida boidinii.

Microbodies, designated as peroxisomes because of their enzyme complement, have been isolated from methanol-grown cells of Candida boidinii. Spheroplast lysates were separated on non-continuous Ficoll density gradients, resulting in a mitochondrial fraction and a peroxisome fraction. Estimates of purity using the mitochondrial enzyme markers suggested that the contamination of mitochondria in the peroxisome fraction was about 2-3%. As shown by electron microscopy the peroxisomes were 0.4-0.6 mum in diameter and contained crystalloid inclusions. Alcohol oxidase and catalase, which catalyse the oxidation of methanol to formaldehyde in Candida boidinii, could be localized within the peroxisomes. Gel-electrophoretic studies of the peroxisome fraction demonstrated that it contained only two predominant protein bands consistent with alcohol oxidase and catalase. No alcohol oxidase and catalase activity was found in mitochondria.     

Catalase, a target of glycation damage in rat liver mitochondria with aging

Aging is characterized by progressive decline of major cell functions, associated with accumulation of altered macromolecules, particularly proteins. This deterioration parallels age-related dysfunction of mitochondria, thought to be a major determinant of this decline in cell function, since these organelles are both the main sources of reactive oxygen species and targets for their damaging effects. To investigate the link between glycation damages that accumulate with aging and the status of mitochondrial antioxidant enzymes, we identified, by mass spectrometry after two dimensional-gel electrophoresis and western blotting, advanced glycation end product-modified matrix proteins in rat liver mitochondria. Catalase appeared to be the only antioxidant enzyme markedly glycated in old rats. Immunogold labeling performed on isolated mitochondria confirmed the mitochondrial matrix location of this enzyme. The content of catalase protein in mitochondrial extract increased with aging whereas the catalase activity was not significantly modified, in spite of a significant increase rate of glycation. Treatment of catalase with the glycating agent fructose led to significant time-dependent inactivation of the enzyme, while methylglyoxal had no noticeable effect. Catalase was co-identified with unglycated glutathione peroxidase-1 in the mitochondrial extracts. Taken together, these results indicate that both anti-oxidant enzymes catalase and glutathione peroxidase-1 housed in liver mitochondria, exhibited a differential sensitivity to glycation; moreover, they lend support to the hypothesis that glycation damages targeting catalase with aging may severely affect its activity, suggesting a link between glycation stress and the age-related decline in antioxidant defense in the mitochondria.     

Efficient Expression of Peroxidatic Activity of Catalase in the Coupled System with Glucose Oxidase—a model of Yeast Peroxisomes

Catalase functioned exclusively to degrade hydrogen peroxide in a reaction mixture containing methanol and hydrogen peroxide, while, when the enzyme was coupled with glucose oxidase, successful conversion of methanol to formaldehyde occurred at the optimized ratio of glucose oxidase to catalase: activity, 1.0 × 10 ^?3; number of molecules, 1.3; protein content, 1. These values in the coupled system were very similar to the ratio of alcohol oxidase to catalase in peroxisomes, one of the subcellular organelles from a methanol-assimilating yeast, Kloeckera sp. 2201, in which these enzymes were coupled to metabolize methanol efficiently. The presence of the optimum ratio in the coupled system in vitro was confirmed by the kinetic analysis of the expression of the peroxidatic activity of catalase coupled with glucose oxidase. Construction of the immobilized system of the coupled enzymes at the optimum ratio demonstrated that the oxidation of methanol through the peroxidatic function of catalase could be continuously and stably operated, the results indicating the usefulness of the system as a model of yeast peroxisomes. Thus, the coupled reaction with glucose oxidase brought out the latent function of catalase, which could not be expected in the system including only catalase.     

Biochemical and molecular characterization of mitochondrial membrane-bound arginase in <Emphasis Type="Italic">Heteropneustes fossilis</Emphasis>

The two predominant forms of arginase, cytosolic Arginase-I and mitochondrial Arginase-II, catalyze hydrolysis of arginine into ornithine and urea. Based on presence of arginase activity in extracts using potassium chloride (KCl), mitochondrial membrane-bound arginase has also been suggested. However, the activity of arginase in fractions obtained after KCl-treatment may be either due to leakage of mitochondrial arginase or release of adhered cytosolic arginase to cell organelles having altered net charge. Therefore, it has been intended to analyse impact of KCl on ultra-structural properties of mitochondria, and biochemical analysis of mitochondrial membrane-bound proteins and arginase of Heteropneustes fossilis. Liver of H. fossilis was used for isolating mitochondria for analysis of ultrastructural properties, preparing cytosolic, mitochondrial, and mitochondrial-membrane bound extracts after treatment of KCl. Extracts were analysed for arginase activity assay, protein profiling through SDS-PAGE and MALDI MS/MS. The KCl-mediated modulation in polypeptides and arginase were also evaluated by PANTHER, MitoProt and IPSORT servers. The effects of KCl on ultra-structural integrity of mitochondria, activity of arginase, modulation on mitochondrial proteins and enzymes including arginase were observed. The 48 kDa polypeptide of mitochondrial fraction, that showed KCl-dependent alteration matched with Myb binding protein and 30 kDa bands resembles to arginase after MALDI MS/MS analysis. Results indicate KCl-dependent ultrastructural changes in mitochondria and release of mitochondrial arginase. The proposed membrane bound mitochondrial arginase could be mitochondrial arginase-II or altered form of cytosolic arginase-I contributing to KCl-induced arginase activity in H. fossilis.     

Alteration of free radical metabolism in the brain of mice infected with scrapie agent.

Alteration of free radical metabolism in the mouse brain by scrapie infection was evaluated. The infection of mice with scrapie agent, 87V strain, slightly increased the activities of catalase and glutathione-S-transferase, while it had no effect on glutathione peroxidase, glutathione reductase, and Cu, Zn-superoxide dismutase. Results show that the scrapie infection decreased the activity of mitochondrial Mn-superoxide dismutase by 50% but increased that of monoamine oxidase (p < 0.05). Scrapie infection also increased the rate of mitochondrial superoxide generation (p < 0.05). Following scrapie infection, the level of free-sulfhydryl compounds in brain homogenates slightly decreased, but the content of thiobarbituric-acid-reactive substances and malondialdehyde increased significantly. Electron microscopy indicated that the ultrastructure of mitochondria was destroyed in the brain of scrapie-infected mice. These results suggest that elevated oxygen free radical generation and lowered scavenging activity in mitochondria might cause the free radical damage to the brain. Such deleterious changes in mitochondria may contribute to the development of prion disease.     

Cytochemical localization of catalase activity in methanol-grown Hansenula polymorpha

The localization of peroxidase activity in methanol-grown cells of the yeast Hansenula polymorpha has been studied by a method based on cytochemical staining with diaminobenzidine (DAB). The oxidation product of DAB occurred in microbodies, which characteristically develop during growth on methanol, and in the intracristate space of the mitochondria. The staining of microbodies was H₂O₂ dependent, appeared to be optimal at pH 10.5, diminished below pH 10 and was inhibited by 20 mM 3-amino 1,2,4 triazole (AT). In contrast to these observations, the reaction in the mitochondria was not H₂O₂ dependent and not notably affected by differences in pH in the range of 8.5 to 10.5. Microbodies and mitochondria were also stained when H₂O₂ was replaced by methanol. Appropriate control experiments indicated that in this case methanol oxidase generated the H₂O₂ for the peroxidative conversion of DAB by catalase. These results suggest that catalase is located in the microbodies of methanol-grown yeasts. A model for a possible physiological function of the microbodies during growth on methanol is put forward.     

The mitochondrial localization of hexokinase in pea leaves

Up to 80% of total cellular hexokinase (EC 2.1.7.4) activity in pea (Pisum sativum L.) leaves was found to be associated with particulate fractions. Fractionation on sucrose density gradients showed this particulate activity to be associated exclusively with mitochondria. In the presence of glucose and ATP, the bound mitochondrial hexokinase could support rates of O₂ uptake of up to 30% of normal ADP-stimulated rates. This stimulation of O₂ uptake by hexokinase was completely sensitive to oligomycin, indicating that it resulted from an increase in the supply of ADP for mitochondrial oxidative phosphorylation. Spectrophotometric measurements of the mitochondrial hexokinase activity showed that ADP could support rapid rates of activity provided oxidizable substrates were also present to support the conversion of ADP to ATP in oxidative phosphorylation. Carboxyatractyloside, an inhibitor of adenine-nucleotide uptake by mitochondria, inhibited this ADP-supported activity, but had no effect on hexokinase activity in the presence of added ATP, demonstrating that the hexokinase enzyme was located external to the inner mitochondrial membrane. Oligomycin also inhibited ADP-supported activity but had no effect on ATP-supported hexokinase activity. Glucose (K_m 53 μM) was the preferred substrate of pea-leaf mitochondrial hexokinase compared with fructose (K_m 5.1 mM). Hexokinase was not solubilised in the presence of glucose-6-phosphate.     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

n-Alkane oxidation byCandida tropicalis

Summary The specific activity of the enzyme catalase was investigated in batch cultures ofCandida tropicalis on the following substrates: Gelsenberg 14/18, n-hexandecane, 1-octadecene, oleyl alcohol, oleic acid and glucose. The catalase activity does not change with the different oxidation levels of the hydrocarbon substrates. A correlation between specific activity and growth rate was established.Inhibition experiments with 2-amino-1,3,4-triazol (AT), a specific catalase inhibitor, gave no evidence for two kinds of hydrogen peroxide-degrading enzymes in yeast cells.Growth on hydrocarbons instead of on glucose enhanced the specific activities of both isocitratelyase (ICL) and catalase to about the same extent.On the other hand the succinate-cytochrome C-oxidoreductase, a mitochondrial enzyme, showed more or less the same activity on both substrates.All these facts suggest that the catalase enzyme is related to the degradation (- or -oxidation) or to the gluconeogenesis (glyoxylate cycle) of fatty acids, but not to the initial oxidation of the alkanes.     

The dual location of β-oxidation enzymes in germinating pea cotyledons

-Oxidation enzymes were detected both in the mitochondria and microbodies of pea cotyledons. Intact mitochondria did not show -oxidation enzyme activity but in ruptured mitochondria this activity was high. It is apparent that the mitochondrial membrane barrier prevents rapid access of acyl-CoA substrates to matrix -oxidation sites. Removal of the membrane barrier permits rapid access of acyl-CoAs and these enzyme activities may then be measured.     

Relative contributions of heart mitochondria glutathione peroxidase and catalase to H(2)O(2) detoxification in in vivo conditions

This study was aimed at assessing the relative contributions to H(2)O(2) detoxification by glutathione peroxidase and catalase in the mitochondrial matrix of heart. For this purpose, mitoplasts from rat heart were used in order to minimize contamination with microperoxisomes, and the kinetic rate constants of both enzymatic activities were determined along with a simulation profile. Results show that the contribution of catalase to H(2)O(2) removal in heart mitochondria is not significant, even under strong oxidative conditions, such as those achieved in ischemia-reperfusion and involving extensive glutathione depletion and high H(2)O(2) concentrations. Conversely, maintenance of the steady state levels of H(2)O(2) in the heart mitochondrial matrix seems to be the domain of glutathione peroxidase. It is suggested that the physiological role of the low amounts of catalase found in heart mitochondria is related to its peroxidatic rather than catalatic activity.     

Nuclear and mitochondrial revertants of a mitochondrial mutant with a defect in the ATP synthetase complex

Summary Yeast strain 990 carries a mutation mapping to the oli1 locus of the mitochondrial genome, the gene encoding ATPase subunit 9. DNA sequence analysis indicated a substitution of valine for alanine at residue 22 of the protein. The strain failed to grow on nonfermentable carbon sources such as glycerol at low temperature (20°C). At 28°C the strain grew on nonfermentable carbon sources and was resistant to the antibiotic oligomycin. ATPase activity in mitochondria isolated from 990 was reduced relative to the wild-type strain from which it was derived, but the residual activity was oligomycin resistant. Subunit 9 (the DCCD-binding proteolipid) from the mutant strain exhibited reduced mobility in SDS-polyacrylamide gels relative to the wild-type proteolipid. Ten revertant strains of 990 were analyzed. All restored the ability to grow on glycerol at 20°C. Mitotic segregation data showed that eight of the ten revertants were attributable to mitochondrial genetic events and two were caused by nuclear events since they appeared to be recessive nuclear suppressors. These nuclear mutations retained partial resistance to oligomycin and did not alter the electrophoretic behavior of subunit 9 or any other ATPase subunit. When mitochondrial DNA from each of the revertant strains was hybridized with an oligonucleotide probe covering the oli1 mutation, seven of the mitochondrial revertants were found to be true revertants and one a second mutation at the site of the original 990 mutation. The oli1 gene from this strain contained a substitution of glycine for valine at residue 22. The proteolipid isolated from this strain had increased electrophoretic mobility relative to the wild-type proteolipid.Abbreviations DCCD dicyclohexylcarbodiimide - SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - SMP submitochondrial particles - mit^- mitochondrial point mutant     

Quantitative immunocytochemical studies on differential induction of serine

Summary Differential induction of serine: pyruvate aminotransferase (SPT) in rat liver parenchymal cells by administration of glucagon or di-(2-ethylhexyl)phthalate (DEHP) was studied using post-embedding immunocytochemical techniques and morphometric methods. Two groups of rats were fasted for 5 days and daily received peritoneal injection of glucagon (300 g/100 g) or physiological saline. Another two groups of rats were fed on laboratory chow with or without 2% DEHP for 2 weeks. Livers were perfusionfixed, cut into tissue sections (50–100 ), and processed to cytochemistry for catalase, immunocytochemistry for SPT, and conventional procedures for electron microscopy. The morphometric analysis showed that glucagon injection has negligible effect on the volume and numerical density and mean diameter of peroxisomes, whereas volume density of mitochondria was decreased by 25%. By DEHP administration peroxisomes were about 3-fold increased in the volume and numerical density. Mitochondria was increased about 40% in the numerical density, but unchanged in the volume density. Light and electron microscopic immunocytochemistry demonstrated that glucagon injection exclusively enhanced mitochondrial SPT, whereas DEHP administration exclusively induced in peroxisomal SPT. Quantitative analysis showed that by the glucagon injection, the labeling density of mitochondria was increased about 4-fold, but that of peroxisomes was 1.6 times as much as control, while by DEHP administration, the labeling density of peroxisomes was enhanced about 3-fold but that of mitochondria was decreased by 13%. The results clearly indicate that glucagon induces mitochondrial SPT, whereas peroxisome proliferator, DEHP induces peroxisomal SPT.     

Induction of Catalase Activity in a Methanol-utilizing Yeast,Kloeckera sp. No. 2201

The methanol-grown cells of Kloeckera sp. No. 2201 exhibit a markedly high catalase activity as compared with the glucose-grown and ethanol-grown cells. In this connection, specific organelles (“microbodies”) appear only in the methanol-grown cells. When the yeast cells harvested from a methanol medium (cells whose catalase activity had been enhanced to an appreciable extent: “partially induced cells”) were transferred into media containing glucose, ethanol or methanol as the sole carbon and energy source, further increase of catalase activity was mediated only by methanol. This induction of catalase activity was partially inhibited by cycloheximide at its high concentration, but chloramphenicol did not show any effect. Glucose inhibited strongly the induction by methanol, while galactose gave no effect. Electron microscopical observation revealed that the development of microbodies in the cells growing on methanol was hardly affected by cycloheximide. Disappearance of microbodies was observed electron microscopically after the methanol-grown cells (partially induced cells) were transferred to a methanol-glucose medium and cultivated for 8 hr. 3′,5′-Cyclic AMP or dibutyryl-3′,5′-cyclic AMP could not eliminate the inhibitory effect of glucose on the catalase induction. Addition of caffeine or theophylline did not promote the action of the cyclic nucleotides. 3-Amino-1,2,4-triazole inhibited only 40% of the hydrogen peroxide-decomposing activity in the cell homogenate of methanol-grown cells even at its concentration of as high as 10 mm, while sodium azide inhibited the enzyme activity completely at the concentration of 1 mm.     

The subcellular localization of glutamate dehydrogenase (GDH): is GDH a marker for mitochondria in brain?

Glutamate dehydrogenase (GDH, EC 1.4.1.2) has long been used as a marker for mitochondria in brain and other tissues, despite reports indicating that GDH is also present in nuclei of liver and dorsal root ganglia. To examine whether GDH can be used as a marker to differentiate between mitochondria and nuclei in the brain, we have measured GDH by enzymatic activity and on immunoblots in rat brain mitochondria and nuclei which were highly enriched by density-gradient centrifugation methods. The activity of GDH was enriched in the nuclear fraction as well as in the mitochondrial faction, while the activities of other mitochondrial enzymes (fumarase, NAD-isocitrate dehydrogenase and pyruvate dehydrogenase complex) were enriched only in the mitochondrial fraction. Immunoblots using polyclonal antibodies against bovine liver GDH confirmed the presence of GDH in the rat brain nuclear and mitochondrial fractions. The GDH in these two subcellular fractions had a very similar molecular weight of 56,000 daltons. The mitochondrial and nuclear GDH differed, however, in their susceptibility to solubilization by detergents and salts. The mitochondrial GDH could be solubilized by extraction with low concentrations of detergents (0.1% Triton X-100 and 0.1% Lubrol PX), while the nuclear GDH could be solubizeded only by elevated concentrations of detergents (0.3% each) plus KCl (>150mM). Our results indicate that GDH is present in both nuclei and mitochondria in rat brain. The notion that GDH may serve as a marker for mitochondria needs to be re-evaluated.     

Adenovirus-mediated overexpression of catalase in the cytosolic or mitochondrial compartment protects against cytochrome P450 2E1-dependent toxicity in HepG2 cells

Cytochrome P450 2E1 (CYP2E1) is an effective producer of reactive oxygen species such as superoxide radical and hydrogen peroxide, which may contribute to the development of alcohol liver disease or cytotoxicity. To investigate the protective role of catalase against CYP2E1-dependent cytotoxicity, E47 cells, a transfected HepG2 cell line overexpressing CYP2E1, were infected with adenoviral vectors containing human catalase cDNA (AdCat) and catalase cDNA with a mitochondrial leader sequence (AdmCat). Forty-eight hours after infection with AdCat or AdmCat at a multiplicity of infection of 100, intracellular catalase protein was increased >2-fold compared with uninfected E47 cells and E47 cells infected with empty adenoviral vector (AdNull) as determined by Western blotting and catalase activity measurements. Overexpression of catalase in the cytosol (AdCat) and in mitochondria (AdmCat) was confirmed by confocal microscopy. Cell death caused by arachidonic acid plus iron was considerably suppressed in both AdCat- and AdmCat-infected E47 cells as determined by assays of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide absorbance, lactate dehydrogenase release, and morphology changes. AdCat- and AdmCat-infected cells were also more resistant to the loss of mitochondrial membrane potential and to the increase in lipid peroxidation induced by arachidonic acid and iron. This study indicates that catalase in the cytosol and catalase in mitochondria are capable of protecting HepG2 cells expressing CYP2E1 against cytotoxicity induced by oxidants that promote lipid peroxidation and suggests the possibility that such agents may be useful in protecting against the development of alcohol liver injury.     

Pyruvate dehydrogenase complex is inhibited in calcium-loaded cerebrocortical mitochondria

An impairment of mitochondrial functions as a result of Ca-loading may be one of the significant events that lead to neuronal death after an ischemic insult. To assess the metabolic consequences of excess Ca on brain mitochondria, pyruvate oxidation was studied in isolated cerebrocortical mitochondria loaded with Ca in vitro. The flux of pyruvate dehydrogenase complex (PDHC) ([1-¹⁴C]pyruvate decarboxylation) was inhibited as the mitochondria accumulated excess Ca under the conditions tested: the inhibition in state 3 (i.e., in the presence of added ADP) was greater than in state 4 (i.e., in the absence of added adenine nucleotides). In state 4, the inhibition of the PDHC flux was accompanied by a similar reduction of the in situ activity of PDHC, indicating a change in PDHC phosphorylation. In state 3, the inhibition of the PDHC flux was greater than the corresponding decrease of the in situ PDHC activity. Thus, mechanisms other than the phosphorylation of PDHC might also contribute to the inhibition of pyruvate oxidation. Measurement of PDHC enzymatic activity in vitro indicated that PDHC, similar to -ketoglutarate dehydrogenase complex, was inhibited by millimolar levels of Ca. This observation suggests that PDHC may also be inhibited non-covalently in Ca-loaded mitochondria in a manner similar to that of -ketoglutarate dehydrogenase complex.