Showing papers on "Catalase published in 1969"

THE SYNTHESIS AND TURNOVER OF RAT LIVER PEROXISOMES: I. Fractionation of Peroxisome Proteins

Abstract: Rat liver peroxisomes isolated by density gradient centrifugation were disrupted at pH 9, and subdivided into a soluble fraction containing 90% of their total proteins and virtually all of their catalase, D-amino acid oxidase, L-α-hydroxy acid oxidase and isocitrate dehydrogenase activities, and a core fraction containing urate oxidase and 10% of the total proteins. The soluble proteins were chromatographed on Sephadex G-200, diethylaminoethyl (DEAE)-cellulose, hydroxylapatite, and sulfoethyl (SE)-Sephadex. None of these methods provided complete separation of the protein components, but these could be distributed into peaks in which the specific activities of different enzymes were substantially increased. Catalase, D-amino acid oxidase, and L-α-hydroxy acid oxidase contribute a maximum of 16, 2, and 4%, respectively, of the protein of the peroxisome. The contribution of isocitrate dehydrogenase could be as much as 25%, but is probably much less. After dissolution of the cores at pH 11 , no separation between their urate oxidase activity and their protein was achieved by Sephadex G-200 chromatography.

The peroxisome: a new cytoplasmic organelle

Abstract: The name ‘peroxisome’ designates a special type of cytoplasmic organelle characterized by the association of one or more oxidases which produce hydrogen peroxide with catalase which destroys the hydrogen peroxide. Early work on peroxisomes has been reviewed by de Duve & Baudhuin (1966). The present paper summarizes recent advances in this field.

A Survey of Plants for Leaf Peroxisomes

Abstract: Leaves of 10 plant species, 7 with photorespiration (spinach, sunflower, tobacco, pea, wheat, bean, and Swiss chard) and 3 without photorespiration (corn, sugarcane, and pigweed), were surveyed for peroxisomes. The distribution pattern for glycolate oxidase, glyoxylate reductase, catalase, and part of the malate dehydrogenase indicated that these enzymes exist together in this organelle. The peroxisomes were isolated at the interface between layers of 1.8 to 2.3 m sucrose by isopycnic nonlinear sucrose density gradient centrifugation or in 1.95 m sucrose on a linear gradient. Chloroplasts, located by chlorophyll, and mitochondria by cytochrome c oxidase, were in 1.3 to 1.8 m sucrose. In leaf homogenates from the first 7 species with photorespiration, glycolate oxidase activity ranged from 0.5 to 1.5 mumoles x min(-1) x g(-1) wet weight or a specific activity of 0.02 to 0.05 mumole x min(-1) x mg(-1) protein. Glyoxylate reductase activity was comparable with glycolate oxidase. Catalase activity in the homogenates ranged from 4000 to 12,000 mumoles x min(-1) x g(-1) wet weight or 90 to 300 mumoles x min(-1) x mg(-1) protein. Specific activities of malate dehydrogenase and cytochrome oxidase are also reported. In contrast, homogenates of corn and sugarcane leaves, without photorespiration, had 2 to 5% as much glycolate oxidase, glyoxylate reductase, and catalase activity. These amounts of activity, though lower than in plants with photorespiration, are, nevertheless, substantial. Peroxisomes were detected in leaf homogenates of all plants tested; however, significant yields were obtained only from the first 5 species mentioned above. From spinach and sunflower leaves, a maximum of about 50% of the marker enzyme activities was found to be in these microbodies after homogenization. The specific activity for peroxisomal glycolate oxidase and glyoxylate reductase was about 1 mumole x min(-1) x mg(-1) protein; for catalase. 8000 mumoles x min(-1) x mg(-1) protein, and for malate dehydrogenase, 40 mumoles x min(-1) x mg(-1) protein. Only small to trace amounts of marker enzymes for leaf peroxisomes were recovered on the sucrose gradients from the last 5 species of plants. Bean leaves, with photorespiration, had large amounts of these enzymes (0.57 mumole of glycolate oxidase x min(-1) x g(-1) tissue) in the soluble fraction, but only traces of activity in the peroxisomal fraction. Low peroxisome recovery from certain plants was attributed to particle fragility or loss of protein as well as to small numbers of particles in such plants as corn and sugarcane. Homogenates of pigweed leaves (no photorespiration) contained from one-third to one-half the activity of the glycolate pathway enzymes as found in comparable preparations from spinach leaves which exhibit photorespiration. However, only traces of peroxisomal enzymes were separated by sucrose gradient centrifugation of particles from pigweed. Data from pigweed on the absence of photorespiration yet abundance of enzymes associated with glycolate metabolism is inconsistent with current hypotheses about the mechanism of photorespiration. Most of the catalase and part of the malate dehydrogenase activity was located in the peroxisomes. Contrary to previous reports, the chloroplast fractions from plants with photo-respiration did not contain a concentration of these 2 enzymes, after removal of peroxisomes by isopycnic sucrose gradient centrifugation.

THE SYNTHESIS AND TURNOVER OF RAT LIVER PEROXISOMES II. Turnover of Peroxisome Proteins

Abstract: After preliminary experiments had established that the injection of Triton WR-1339 necessary for the separation of lysosomes and peroxisomes did not affect the turnover rate of catalase, the decay of 3H-leucine incorporated into peroxisomes was studied in whole particles and in protein subfractions. It was shown that peroxisomes are destroyed in a completely random way, probably as wholes since the apparent half-life was the same for all subfractions, about 3½ days. In agreement with the results of Price et al. (11), the half-life of catalase derived from the rate of recovery from aminotriazole inhibition was about 11½ days, as was the apparent half-life of the heme prosthetic groups measured with 14C-α-aminolevulinic acid. Guanidino-labeled arginine gave an apparent half-life of 2½ days with large statistical uncertainty. Either the leucine label was reutilized very extensively in our animals and the true half-life of peroxisomes is 1½ days, or the prosthetic groups of catalase turn over more rapidly than the protein part of the molecule.

The use of beef liver catalase as a protein tracer for electron microscopy

Abstract: Beef liver catalase was injected intravenously into mice, and its distribution in the kidney, myocardium, and liver was studied with the electron microscope A specific and relatively sensitive method was developed for its ultrastructural localization, based on the peroxidatic activity of catalase and employing a modified Graham and Karnovsky incubation medium The main features of the medium were a higher concentration of diaminobenzidine, barium peroxide as the source of peroxide, and pH of 85 Ultrastructurally, the enzyme was seen to permeate the endothelial fenestrae and basement membranes of tubular and glomerular capillaries of the kidney The urinary space and tubular lumina contained no reaction product In the myocardial capillaries, the tracer filled the pinocytotic vesicles but did not diffuse across the intercellular clefts of the endothelium In liver, uptake of catalase was seen both in hepatocytes and in Kupffer cells

Catalase-Aminotriazole Method for Measuring Secretion of Hydrogen Peroxide by Microorganisms

Abstract: A new method for measuring the secretion of H(2)O(2) has been based upon an H(2)O(2)-dependent inhibition of catalase by 3-amino-1,2,4-triazole. The conversion of an H(2)O(2)-secretion rate into a catalase inhibition rate amplified a relatively small molar concentration of H(2)O(2) and provided a highly specific and sensitive method for quantitatively measuring H(2)O(2). A major advantage of this approach is that it does not require extensive accumulation of H(2)O(2) in the environment. The method was successfully employed to measure H(2)O(2) secretion by Mycoplasma pneumoniae, which possesses a peroxidase-like activity that limits the accumulation of H(2)O(2) in the environment.

Antistreptococcal Activity of Lactoperoxidase

Abstract: A study of the inhibition of the growth of Streptococcus cremoris 972 by the enzyme lactoperoxidase has shown, in agreement with previous investigations, that the inhibition requires a source of both peroxide and thiocyanate. The thiocyanate may play more than one role. It stabilizes the very dilute solutions of lactoperoxidase employed in these studies, and its oxidation products may be involved in the inhibition. Binding of the enzyme by the microorganism is suggested by the fact that when the organism was preincubated with the enzyme and then in a medium free from the enzyme, but containing peroxide and thiocyanate, the growth of the organism was inhibited. This inhibition has all the properties of the enzyme-containing system. Although no dialyzable factor could be demonstrated to cause the inhibition, the inhibitory state involving peroxide, the enzyme, and thiocyanate survived for at least 60 min before cells were added to the medium. When catalase was present in the medium prior to the addition of the cells, the inhibition was completely reversed. It was only partially reversed if catalase was added a few moments after the addition of the cells. The data have been interpreted as indicating that the inhibition takes place rapidly and requires the formation of a quaternary complex of the cells, thiocyanate, peroxide, and the enzyme lactoperoxidase.

Biological Response of Lactic Streptococci and Lactobacilli to Catalase

Abstract: Addition of catalase to milk cultures of lactic streptococci resulted in increased rates of acid production, although it had no effect on cultures of lactobacilli. Milk cultures of both streptococci and lactobacilli produced detectable amounts of peroxide, which reached a maximum level in the early period of acid production followed by a drastic decrease as the acid production increased. Pyruvate and reduced glutathione decreased the amount of peroxide formed, but had little effect on acid production by the streptococci. Ferrous sulfate prevented the accumulation of peroxide and stimulated the rate of acid production by the streptococci to a greater extent than did catalase.

Role of the intracellular distribution of hepatic catalase in the peroxidative oxidation of methanol

Abstract: Previous to 1950, when Bonnichsenl isolated crystalline alcohol dehydrogenase (ADH) and showed that it did not react with methanol,$ it was believed that the ADH system was responsible for the oxidation of all primary aliphatic alcohols, including methanol (FIGURE 1 ) . This observation redirected attention to the peroxidative system as a means of oxidizing methanol. As early as 1936, Keilin and Hartree3 showed that catalase catalyzed the oxidation of alcohols to their aldehydes when hydrogen peroxide was supplied in low concentrations, as might be provided in living cells through the action of flavin and other peroxide-generating enzymes. They presented arguments to support their view that catalase is not present in the tissues to protect against peroxide intoxication, as was widely contended, but rather to carry out coupled (peroxidative) oxidations. Employing techniques that permitted very rapid spectral determinations, Chance4 identified the intermediate complexes and analyzed the kinetics of the components of the reaction (FIGURE 1 ) . Ideas were exchanged for several years as to whether or not the peroxidative system participated in the in vivo oxidation of methanol and other alcohols. A direct means of examining the question was provided when Heim and coworkers5 showed that the intraperitoneal injection of 3-amino-1,2,4-triazole (AT) caused a reduction in hepatic and renal catalase activities of 90% or more. Mannering and Parkss found that the AT-induced inhibition of hepatic catalase was accompanied by a 70% reduction of the methanol-oxidizing capacity of rat liver homogenates. The addition of crystalline beef liver catalase to these homogenates restored methanol oxidation to normal. While these in vitro studies pointed to a role of catalase in methanol oxidation, they did little to establish its participation in vivo. Because of the complexity of the system, involving as it does the rate of formation of hydrogen peroxide, which in turn depends upon the concentration of substrates available to the peroxide-generating enzymes as well as the availability of hepatic catalase to the hydrogen peroxide produced by these enzymes, it seemed unlikely that in vitro studies would provide much information as to what was occurring in the intact animal. This presentation is devoted largely to a review of the evidence that shows that the intact rat oxidizes methanol largely through the peroxidative system, but that ethanol oxidation proceeds differently, probably almost entirely via the ADH system. The evidence is based on in vitro studies that employed several approaches: 1 ) 14C-methanol and 1 -Wethano1 oxidation were studied

Relationship of Hydrogen Peroxide Production by Mycoplasma pulmonis to Virulence for Catalase-deficient Mice

Abstract: Mycoplasma pulmonis, an etiological agent of murine pneumonia, produced about 0.065 mumoles of hydrogen peroxide (H(2)O(2)) per hr per 10(10) colony-forming units. When glucose was present at a concentration of 0.01 m, H(2)O(2) production was increased by 50%. To determine if H(2)O(2) production by M. pulmonis could be correlated with virulence, normal, acatalasemic, and acatalatic mice were infected with the organism. Three days after infection with M. pulmonis significantly more acatalatic mice had pneumonia than did normal or acatalasemic mice. The pneumonia in acatalatic mice was also more severe than in the other two groups. Five days after infection, pneumonia in the acatalatic mice was resolved, whereas normal mice were severely affected. The presence of pneumonia and the severity were correlated with the recovery of M. pulmonis from the lesions. In vitro studies of the effect of catalase on M. pulmonis showed that exogenously supplied catalase stimulated the growth of M. pulmonis at 37 C and prolonged its survival at 25 C. Hemolysis of sheep blood, guinea pig blood, rabbit blood, and normal and acatalasemic mouse blood by M. pulmonis was inversely related to the catalase activity of the erythrocytes. These findings suggest that H(2)O(2) secretion contributes to the virulence of M. pulmonis and to the death of the microorganism in the absence of host catalase.

Urate Oxidase and its Association with Peroxisomes in Acanthamoeba sp.

Abstract: Urate oxidase is present in axenic Acanthamoeba sp. (Neff strain). The enzyme shows an absolute requirement for molecular oxygen as electron acceptor and forms hydrogen peroxide. Its optimum of activity in 0.1 M Tris-glycine buffer is at pH 9.4 with an apparent Km value of 18.7 μM. Its activity is inhibited by oxypurines, trichloropurine, oxonate and potassium cyanide. In all these properties the enzyme strongly resembles mammalian urate oxidases. Isopycnic centrifugation of Acanthamoeba homogenates in sucrose gradients showed that the distribution pattern of urate oxidase is identical with that of catalase but is different from the distribution of succinate dehydrogenase (a marker enzyme for mitochondria) and of acid phosphatase (a marker enzyme for lysosomes). The results strongly suggest the existence of a particle population with characteristics of peroxisomes in Acanthamoeba.

Antimicrobial Activity of Gatalase at Acid pH

Abstract: SummaryCrystalline catalase exerts an antimicrobial effect when combined with iodide ions and a H2O2 generating system. The antimicrobial effect is optimal at pH levels below 5.0 and is inhibited by azide, cyanide, ascorbic acid, reduced glutathione, cysteine, 3-amino-1,2,4-triazole, methimazole, propylthiouracil, ergothioneine, thiocyanate, thiosulfate, formate, ethanol, NADH, NADPH, and tyrosine. The influence of catalase, of either leukocytic or microbial origin, on the antimicrobial activity of the leukocyte is considered.

Light microscopic study of the peroxidatic activity of catalase in formaldehyde-fixed rat liver.

Abstract: The peroxidatic activity of rat liver catalase was demonstrated by histochemical staining with 3,3'-diaminobenzidine as hydrogen donor. The activity was so weak that its location was hard to identify in formaldehyde-fixed cells, although high catalatic activity was present, as evidenced by the production of bubbles upon the addition of hydrogen peroxide to the incubation medium. Pretreatment of fixed sections for 60 min at 37°C with formamide, urea or trypsin enhanced the peroxidatic activity significantly. The reaction granules scattered throughout the cytoplasm of the parenchymal cells probably correspond to the peroxisomes.

Immunohistochemical localization of catalase in mammalian tissues.

Abstract: The distribution of catalase was investigated in bovine tissues using fluorescent antibody techniques. The immunochemical properties of liver catalase were also examined. Two distinct components of liver catalase in immune system were found. Both of them possessed common antigenicity with erythrocyte catalase. No cross-reactivity was observed by immunodiffusion between catalase and the other hemoproteins such as lactoperoxidase, cytochrome c and hemoglobins. Bovine liver, pancreas, kidney, spleen and peripheral blood were examined. Catalase was located mainly in the cytoplasm of hepatic cells, acinar cells of the pancreas, epithelia of proximal tubuli, splenic cells scattered in the red pulp and some leukocytes. It was not found in any nucleus. Intracorpuscular catalase could be revealed in the erythrocytes treated with surface-active agents but not in frozen sections.

Glucose-dependent secretion and destruction of hydrogen peroxide by Mycoplasma pneumoniae.

Abstract: The secretion of H(2)O(2) by Mycoplasma pneumoniae and M. gallisepticum was measured with the new catalase-aminotriazole method. Peroxide secretion by the mycoplasmas was stimulated by glucose. When catalase and aminotriazole were omitted and exogenous H(2)O(2) was added to the mycoplasmas, a loss in H(2)O(2) was noted with time; the addition of glucose speeded the disappearance of H(2)O(2). The presence of this peroxidase-like activity in the mycoplasmas explains an observed failure of H(2)O(2) to accumulate freely in the suspension medium.

Action of Hydrogen Peroxide on Growth Inhibition of Salmonella typhimurium

Abstract: SUMMARY: The effect of hydrogen peroxide on the growth of Salmonella typhimurium lt2 in a mineral glucose medium was investigated. The H2O2 produced a lag period, the duration of which increased as the concentration of H2O2 in the medium was increased from 1 to 60 μg./ml. Growth subsequent to the lag period proceeded at the normal growth rate at concentrations of H2O2 as high as 30 μg./ml. Storage of H2O2 in the sterile growth medium resulted in a disappearance of H2O2 with a half-life of about 48 hr. The disappearance of H2O2 because of reaction with glucose resulted in proportionate decreases in the growth inhibitory action of the medium. Salmonella typhimurium destroyed H2O2 rapidly (half-time = 60 min.)—an effect attributed largely to bacterial catalase. The catalatic activity of the bacteria was decreased or eliminated by boiling or by treatment with cyanide. The growth of the bacteria in H2O2 resulted in the development of H2O2 resistance. By subculturing the wild type lt2 in medium containing successively higher H2O2 concentration, a resistant strain designated lt2 p was isolated which grew in the presence of H2O2 concentrations that were completely inhibitory to the wild type. The experimental results support the generally accepted conclusion that bacterial catalase acts in protecting the organism from H2O2. It is pointed out that in biological media H2O2 readily forms adducts with many compounds, including carbonyls, amino acids and thymine. Consequently, the inhibitory effects of H2O2, especially in studies involving auxotrophs, may be partly or wholly due to the peroxide adduct rather than ‘free’ H2O2.

Apparatus for detection of catalase-containing bacteria

Abstract: SENSITIVE FAST-RESPONSE APPARATUS IS DESCRIBED FOR THE DETECTION OF THE PRESENCE OF (A) AEROBIC OR FACULTATIVELY ANAEROBIC BACTERIA, (B) CERTAIN BODY CELL BREAKDOWN PRODUCTS IN BODY FLUIDS OR (C) UNUSUAL CONTAMINATION OF THE AMBIENT BY AEROBIC BACTERIA. THE METHOD EMPLOYED WHICH ENABLES DYNAMIC, CONTINUOUS OBSERVATION OF CATALYASEH2O2 REACTION, IS BASED UPON MEASURING THE INCREMENTAL INCREASE OF OXYGEN PARTIAL PRESSURE WITHIN A HYDROGEN PEROXIDE SOLUTION. THE INHERENT PRESSURE WITHIN A HYDROGEN BACTERIA AND IN MOST ANIMAL CELL PROMOTES THE RAPID DECOMPOSITION OF HYDROGEN PEROXIDE RESULTING IN THE LIBERATION OF OXYGEN. THIS MEASUREMENT OF OXYGEN PARTIAL PRESSURE IS MADE DIRECTLY FROM THE HYDROGEN PEROXIDE SOLUTION BY MEANS OF AN OXYGEN PERMEABLE MEMBRANE POLAROGRAPHIC CELL EQUIPPED WITH A CATHODE CONFIGURATION PROVIDING IMPROVED SENSITIVITY. THE PRESENCE OF SMALL NUMBERS OF BACTERIA OR OTHER CATALASE-CONTAINING CELLS MAY BE QUANTITATIVELY DETERMINED BY CONDUCTING THE CATALASEH2O2 REACTION IN A VERY SMALL REPRODUCIBLE REACTION VOLUME.

Inhibition of catalase in perfused rat liver by sodium azide.

Abstract: In spite of intensive studies of the mechanism of action of catalase by Chance and Theorell and their coworkers, its role in vivo, if any, is Whether it acts predominantly peroxidatically (requiring hydrogen donors such as ethanol or formate and producing water) or catalatically (producing oxygen and water) in vivo has remained obscure. Portwich and Abei demonstrated a coupling of oxygen uptake to formate oxidation and suggested a peroxidatic role for catalase in rat liver slices.4 On the other hand, bacterial catalase was shown by Chance to react “catalatically” in the intact cells of Micrococcus lysodeik t ic~s .~ deDuve and Baudhuin observed that most of the catalase in liver is located within the peroxisome? They also proposed that reducing equivalents generated in the extramitochondrial space may be oxidized peroxidatically via catalase in the peroxisome, obviating the necessity to postulate substrate shuttle mechanisms between the extraand intramitochondrial spaces under some metabolic conditions. This communication reports experiments in which hydrogen peroxide increased and sodium azide decreased effluent oxygen concentrations from the perfused rat liver, indicating that exogenously added, as well as endogenously generated, peroxide reacts catalatically in that tissue. These data, together with calculations on the ratio of exogenous hydrogen donor to hydrogen peroxide concentrations necessary to activate the peroxidatic reaction, and the fact that no known substrate for the reaction exists at adequate concentrations in normal, metabolizing rat liver, leads to the conclusion that catalase reacts predominantly catalatically in vivo in rat liver. Methods

PHOTOINACTIVATION OF CATALASES FROM MAMMAL LIVER, PLANT LEAVES AND BACTERIA. COMPARISON OF INACTIVATION CROSS SECTIONS AND QUANTUM YIELDS AT 406 nm

Abstract: — Photoinactivation in vitro at pH 70 of catalases from different sources (bovine liver, spinach leaves, and Micrococcus lysodeikticus) was studied The wavelength of the inactivating light was close to the Soret peak of catalase No great difference in light sensitivity between soluble catalases were found; the inactivation cross sections found ranged from 3810-4 to 50 10-4A2/molecule The inactivation quantum yield is 22 10-5 for bovine liver catalase and 3110-5 for Micrococcus catalase The quantum yield for soluble spinach catalase is of a similar order of magnitude There are some indications of a greater resistance to photodestruction of the spinach leaf catalase activity associated with small particles

Organic Peroxides and the Antibacterial Action of Irradiated Sucrose as Affected by Catalase

Abstract: Sucrose solutions irradiated in the presence or absence of oxygen inhibit the growth of Salmonella typhimurium. The addition of catalase prior to or shortly after inoculation of the organisms into ...