Showing papers on "Catalase published in 1973"

Induction of Superoxide Dismutase by Molecular Oxygen

Abstract: Oxygen induces superoxide dismutase in Streptococcus faecalis and in Escherichia coli B. S. faecalis grown under 20 atm of O2 had 16 times more of this enzyme than did anaerobically grown cells. In the case of E. coli, changing the conditions of growth from anaerobic to 5 atm of O2 caused a 25-fold increase in the level of superoxide dismutase. Induction of this enzyme was a response to O2 rather than to pressure, since 20 atm of N2 was without effect. Induction of superoxide dismutase was a rapid process, and half of the maximal level was reached within 90 min after N2-grown cells of S. faecalis were exposed to 20 atm of O2 at 37 C. S. faecalis did not contain perceptible levels of catalase under any of the growth conditions investigated by Stanier, Doudoroff, and Adelberg (23), and the concentration of catalase in E. coli was not affected by the presence of O2 during growth. S. faecalis, which had been grown under 100% O2 and which therefore contained an elevated level of superoxide dismutase, was more resistant of 46 atm of O2 than were cells which had been grown under N2. E. coli grown under N2 contained as much superoxide dismutase as did S. faecalis grown under 1 atm of O2. The E. coli which had been grown under N2 was as resistant to the deleterious effects of 50 atm of O2 as was S. faecalis which had been grown under 1 atm of O2. These results are consistent with the proposal that the peroxide radical is an important agent of the toxicity of oxygen and that superoxide dismutase may be a component of the systems which have been evolved to deal with this potential toxicity.

Oxygen Toxicity and the Superoxide Dismutase

Abstract: Oxygen caused an increase in the amount of superoxide dismutase in Escherichia coli B but not in Bacillus subtilis. E. coli B cells, induced by growth under 100% O2, were much more resistant to the lethal effects of 20 atm of O2 than were cells which contained the low uninduced level of this enzyme. In contrast, B. subtilis, which could not respond to O2 by increasing its content of superoxide dismutase, remained equally sensitive to hyperbaric O2 whether grown under 100% O2 or areobically. The catalase in these organisms exhibited a reciprocal response to oxygen. Thus, the catalase of E. coli B was not induced by O2, whereas that of B. subtilis was so induced. These results are consistent with the view that superoxide dismutase is an important component of the defenses of these organisms against the toxicity of oxygen, whereas their catalases are of secondary importance in this respect. The ability of streptonigrin to generate O2−, by a cycle of reduction followed by spontaneous reoxidation, has been verified in vitro. It is further observed that E. coli B which contain the high induced level of superoxide dismutase were more resistant to the lethality of this antibiotic, in the presence of oxygen, than were E. coli B which contained the low uninduced level of this enzyme. This difference between induced and uninduced cells was eliminated by the removal of O2. These results are consistent with the proposal that the enhanced lethality of streptonigrin under aerobic conditions may relate to its in vivo generation of O2− by a cycle of reduction and spontaneous reoxidation. In toto, these observations lend support to the hypothesis that O2− is an important agent of oxygen toxicity and that superoxide dismutase functions to blunt the threat posed by this reactive radical.

Phenol hydroxylase from yeast

Abstract: An enzyme, able to carry out an NADPH-dependent hydroxylation of monocyclic phenols, was purified 20–30-fold from Trichosporon cutaneum grown on phenol or resorcinol as a major carbon source The purified enzyme was homogeneous upon analytical disc electrophoresis The enzyme is a bright-yellow protein with an absorption spectrum typical of flavoproteins Its molecular weight is 148000, and its estimated FAD content is approximately 1 mole per mole enzyme The purified enzyme has essentially the same broad substrate specificity as crude preparations In addition to phenol it also hydroxylates the three isomeric diphenols and a number of phenol derivatives to their corresponding o-diols Km values for the three substrates of the holoenzyme are all of the order 10 μM : Km (phenol) = 18 μM, Km(NADPH) = 71 μM The absorption spectrum of the holoenzyme is modified in the presence of phenol FAD could be resolved from the purified enzyme by preparative disc electrophoresis Reassembly of the holoenzyme required SH-groups The enzyme activity is unaffected by chelators of iron and copper but it is inhibited by heavy metals The inhibition by p-chloromercuribenzoate is readily reversed by dithiothreitol Among oxidizing agents, hydrogen peroxide and peroxidase depressed the enzyme activity, whereas catalase was without effect Among reducing agents ascorbate depressed enzyme activity Sodium dithionite and sodium borohydride bleached the enzyme with concomitant loss of activity After reduction with dithionite, the enzyme was rapidly re-oxidized, re-gaining its original activity After reduction with borohydride re-oxidation was very slow However, the enzyme could be re-activated by incubation with FAD The enzyme is very sensitive to inorganic salts, nitrogen bases and detergents Chloride is quite deleterious whereas phosphate seems to stabilize the enzyme

The characteristics of the "peroxidatic" reaction of catalase in ethanol oxidation.

Abstract: Ethanol oxidation by rat liver catalase (the ‘peroxidatic’ reaction) was studied quantitatively with respect to the rate of H2O2 generation, catalase haem concentration, ethanol concentration and the steady-state concentration of the catalase–H2O2 intermediate (Compound I). At a low ratio of H2O2-generation rate to catalase haem concentration, the rate of ethanol oxidation was independent of the catalase haem concentration. The magnitude of the inhibition of ethanol oxidation by cyanide was not paralleled by the formation of the catalase–cyanide complex and was altered greatly by varying either the ethanol concentration or the ratio of the rate of H2O2 generation to catalase haem concentration. The ethanol concentration producing a half-maximal activity was also dependent on the ratio of the H2O2-generation rate to catalase haem concentration. These phenomena are explained by changes in the proportion of the ‘catalatic’ and ‘peroxidatic’ reactions in the overall H2O2-decomposition reaction. There was a correlation between the proportion of the ‘peroxidatic’ reaction in the overall catalase reaction and the steady-state concentration of the catalase–H2O2 intermediate. Regardless of the concentration of ethanol and the rate of H2O2 generation, a half-saturation of the steady state of the catalase–H2O2 intermediate indicated that about 45% of the H2O2 was being utilized by the ethanol-oxidation reaction. The results reported show that the experimental results in the study on the ‘microsomal ethanol-oxidation system’ may be reinterpreted and the catalase ‘peroxidatic’ reaction provides a quantitative explanation for the activity hitherto attributed to the ‘microsomal ethanol-oxidation system’.

Metabolic alterations produced in the liver by chronic ethanol administration. Increased oxidative capacity.

Abstract: 1. Administration of ethanol (14g/day per kg) for 21-26 days to rats increases the ability of the animals to metabolize ethanol, without concomitant changes in the activities of liver alcohol dehydrogenase or catalase. 2. Liver slices from rats chronically treated with ethanol showed a significant increase (40-60%) in the rate of O(2) consumption over that of slices from control animals. The effect of uncoupling agents such as dinitrophenol and arsenate was completely lost after chronic treatment with ethanol. 3. Isolated mitochondria prepared from animals chronically treated with ethanol showed no changes in state 3 or state 4 respiration, ADP/O ratio, respiratory control ratio or in the dinitrophenol effect when succinate was used as substrate. With beta-hydroxybutyrate as substrate a small but statistically significant decrease was found in the ADP/O ratio but not in the other parameters or in the dinitrophenol effect. Further, no changes in mitochondrial Mg(2+)-activated adenosine triphosphatase, dinitrophenol-activated adenosine triphosphatase or in the dinitrophenol-activated adenosine triphosphatase/Mg(2+)-activated adenosine triphosphatase ratio were found as a result of the chronic ethanol treatment. 4. Liver microsomal NADPH oxidase activity, a H(2)O(2)-producing system, was increased by 80-100% by chronic ethanol treatment. Oxidation of formate to CO(2)in vivo was also increased in these animals. The increase in formate metabolism could theoretically be accounted for by an increased production of H(2)O(2) by the NADPH oxidase system plus formate peroxidation by catalase. However, an increased production of H(2)O(2) and oxidation of ethanol by the catalase system could not account for more than 10-20% of the increased ethanol metabolism in the animals chronically treated with ethanol. 5. Results presented indicate that chronic ethanol ingestion results in a faster mitochondrial O(2) consumption in situ suggesting a faster NADH reoxidation. Although only a minor change in mitochondrial coupling was observed with isolated mitochondria, the possibility of an uncoupling in the intact cell cannot be completely discarded. Regardless of the mechanism, these changes could lead to an increased metabolism of ethanol and of other endogenous substrates.

The synthesis and turnover of rat liver peroxisomes. V. Intracellular pathway of catalase synthesis.

Abstract: The subcellular distribution of the biosynthetic intermediates of catalase was studied in the livers of rats receiving a mixture of [3H]leucine and [14C]δ-aminolevulinic acid by intraportal injection. Postnuclear supernates were fractionated by a one-step gradient centrifugation technique that separates the main subcellular organelles, partly on the basis of size, and partly on the basis of density. Labeled catalase and its biosynthetic intermediates were separated from the gradient fractions by immunoprecipitation, and the distributions of radioactivity were compared with those of marker enzymes. The results show that catalase protein is synthesized outside the peroxisomes, but rapidly appears in these particles, mostly still in the form of the first hemeless biosynthetic intermediate. Addition of heme and completion of the catalase molecule take place within the peroxisomes. During the first 15 min after [3H]leucine administration, more than half of the newly formed first intermediate was recovered in the supernatant fraction, where it was found to exist as an aposubunit of about 60,000 molecular weight.

THE SYNTHESIS AND TURNOVER OF RAT LIVER PEROXISOMES : IV. Biochemical Pathway of Catalase Synthesis

Abstract: Early events in the biosynthesis of liver catalase were studied on female rats receiving [ 3 H]leucine or [ 3 H]δ-aminolevulinic acid or a mixture of [ 3 H]leucine with [ 14 C]δ-aminolevulinic acid by intraportal injection. Catalase antigen was selectively separated from homogenates by immunoprecipitation, both without and after partial purification of the enzyme. Label from both precursors appeared first in immunoprecipitable material which was lost upon purification of catalase; the label subsequently became associated with material indistinguishable from catalase. Kinetic analysis of the results indicates that the nonpurifiable material identified by early labeling consists of two distinct biosynthetic intermediates, the first lacking heme and representing about 1.6% of the total catalase content or 13 µg/g liver, the second containing heme and representing about 0.5% of the total catalase content or 4 µg/g liver. The first intermediate migrates at the same rate as catalase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and therefore has a monomeric molecular weight of about 60,000.

Induction of hepatic microsomal reduced nicotinamide adenine dinucleotide phosphate-dependent production of hydrogen peroxide by chronic prior treatment with ethanol.

Abstract: Treatment of rats for 21-28 days with a semiliquid diet containing ethanol resulted in a near doubling of liver microsomal cytochrome P-450 content. Concomitantly, statistically significant increases in the rate of NADPH oxidation, "endogenous" respiration, and acetaldehyde formation from ethanol in microsomes were observed. An average increase in NADPH-dependent hydrogen peroxide formation of 45 ± 7% (SE) was observed as a result of chronic ethanol treatment, employing the decrease in scopoletin fluorescence or the formation of cytochrome peroxidase complex II as hydrogen peroxide-detecting systems. Since it has been reported that the rate-limiting step for ethanol oxidation in microsomes is the rate of generation of hydrogen peroxide for the peroxidatic reaction of catalase [R. G. Thurman, H. G. Ley, and R. Scholz, Eur. J. Biochem. 25, 420-430 (1972)], this adaptive increase in hydrogen peroxide production due to chronic ethanol treatment most likely accounts for the enhanced ethanol oxidation via catalase-H 2 O 2 . The data are consistent with the hypothesis that microsomal ethanol oxidation is due to peroxidation via catalase utilizing microsomal hydrogen peroxide.

Superoxide dismutase: a contaminant of bovine catalase (Short Communication)

Abstract: Some commercial samples of bovine catalase contain superoxide dismutase activity. Therefore the inhibition of a reaction on the addition of a catalase preparation need not necessarily mean that H2O2 is responsible for the reaction.

Novel catalatic proteins of bakers' yeast. I. An atypical catalase.

Abstract: At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

Analysis of the catalase--hydrogen peroxide intermediate in coupled oxidations.

Abstract: In previous communications, we considered the kinetics of the catalase-H202 intermediate in coupled oxidations ofthe isolated liver catalase (Chance, 1949) and of the mixed mitochondrial-peroxisomal fraction (Chance & Oshino, 1971). In those cases, it was possible to record the kinetics of formation of the intermediate by H202 pulses, and solve for the H202 concentration. In most ofthe cases, however, only the analysis of steady-state effects are feasible and will be useful for the further study on the catalase reaction, especially in a system such as the perfused liver. It seems appropriate, therefore, to extend the analysis. The chemical equations for the catalase reactions are:

Untersuchungen zur Funktionsänderung der Microbodies in den Keimblättern von Helianthus annuus L.

Abstract: The enzyme patterns in sunflower cotyledons indicate that the glyoxysomal function of microbodies is replaced by the peroxisomal function of these organelles during the transition from fat degradation to photosynthesis. The separation of the microbody population into glyoxysomes and peroxisomes during this transition period is reported. The mean difference in density between the activity peaks of glyoxysomal and peroxisomal marker enzymes on a sucrose gradient was calculated to be 0.007±0.004 g/cm3 and turned out to be significant (t=7.8>4.04=t 5;0.01). The activity peak of catalase coincides with that of isocitrate lyase in early stages of development, but shifts to the activity peak of peroxisomal marker enzymes during the transition period. No isozymes of the catalase could be detected by gel electrophoresis in the microbodies with the two different functions. During the rise of the peroxisomal marker enzymes no synthesis of the common microbody marker, catalase, could be demonstrated using the inhibitor allylisopropylacetamide. Using D2) for density labeling of newly-formed catalase, no difference is observed between the density of catalase from cotyledons grown on 99.8% D2O during the transition period and the density of enzyme from cotyledons grown on H2O. The activity of particulate glycolate oxidase is reduced 30–50% by allylisopropylacetamide, but is not affected by D2O. The chlorophyll formation in the cotyledons is strongly inhibited by both substances.

The role of hydrogen peroxide and catalase in hepatic microsomal ethanol oxidation.

Abstract: The basic evidence upon which a unique microsomal ethanol-oxidizing system was postulated was the following: a) requirements for NADPH and oxygen; b) partial inhibition by carbon monoxide; c) induction by chronic ethanol pretreatment; and d) different sensitivities of MEOS and catalase to inhibitors (table 5). On the other hand, the requirement for NADPH and oxygen and partial sensitivity to CO are also properties of microsomal hydrogen peroxide formation from NADPH, which is also induced by chronic ethanol pretreatment.5 Moreover, several investigators have failed to differentiate a unique MEOS from catalase based on inhibitor studies [fig. 3; table 2 (6, 216; table 5]. In addition, the inhibition of microsomal ethanol oxidation by formate, azide, and H2O2 utilizing systems and its stimulation by menadione are consistent with a peroxidatic process catalyzed by contaminating catalase which utilizes hydrogen peroxide formed from NADPH. Lastly, an active reconstituted mixed function oxidation system free of catalase activity failed to oxidize ethanol (table 3). Thus, it is our conclusion that the microsomal ethanol-oxidizing system is due to generation of hydrogen peroxide by microsomal components (e.g., NADPH oxidase, cytochrome P-450) and NADPH (fig. 8). Hydrogen peroxide in turn acts with contaminating catalase to convert ethanol into acetaldehyde. As can be seen from a careful examination of table 5, the postulation of a unique MEOS in addition to the peroxidatic reaction of catalase is superfluous.

Hydrogen Peroxide in Erythrocytes

Abstract: Exposure of rats to 5.0 ppm of ozone, and of mice to 6.7 ppm of ozone, for 90 minutes results in a significant decline in erythrocyte catalase levels in animals pretreated with the catalase inhibitor, aminotriazole (ATZ). As ATZ inhibits catalase only after the enzyme has first reacted with hydrogen peroxide, this decline in catalase activity indicates the presence of hydrogen peroxide within erythrocytes during ozone inhalation. Exposure to lower levels of ozone did not produce detectable levels of hydrogen peroxide within erythrocytes. In vitro evaluation of this technique suggests that, as in the human, catalase is not the major enzyme detoxifying hydrogen peroxide in rat erythrocytes. The ATZ-catalase method is therefore limited in its ability to detect the ozone-induced presence of hydrogen peroxide beyond the pulmonary endothelium.

The Effects of γ-irradiation on Solutions of Catalase, Apocatalase and Haematin

Abstract: SummaryDilute aqueous solutions of catalase, apocatalase and haematin were irradiated; the hydroxyl radical was the major inactivating species. The initial destruction of the haem group in the enzyme was related to the loss of catalytic activity. Cysteyl, histidyl and tyrosyl residues were destroyed during the radiolysis of the apocatalase and catalase and protection of the haematin was afforded by the apoenzyme.