Enzymic and morphological studies on catalase positive particles from brown fat of cold adapted rats

Summary Brown adipose tissue of normal and cold-adapted adult rats has been investigated morphologically and cytochemically. In thin sections catalase-positive particles appear as circular, oval or elongated profiles lying either as single particles or forming groups. Biochemical studies on peroxisomal enzymes show an increase of catalase activity to the tenfold amount after cold adaptation. The tissue is devoid of D-aminoacid oxidase and glycolate oxidase, while low activities of middle-chain -hydroxyacid oxidases could be detected. The catalase-positive particles were purified by differential and isopycnic gradient centrifugation. The density of the particles (1.20 g/cm³) is lower than that of the liver peroxisomes. Enzymic investigations of the fractions render it probable that particles contain carnitine acetyltransferase, whereas they are lacking NAD-dependent glycerophosphate dehydrogenase. The pellets derived from the gradient centrifugation have been checked morphologically for purity. After performing DAB-cytochemistry for identification of the peroxidatic activity of catalase, most of the particles were shown to be structurally intact and homogeneously filled with reaction product.     

Catalase-positive particles (“microperoxisomes”) from rat preputial gland and bovine adrenal cortex

Catalase activity was detected histochemically within membrane-bound cell organelles in epithelial cells of rat preputial gland and bovine adrenal cortex. These particles are oval to worm-like in rat preputial gland, 0.08 – 0.15 μm thick and up to 1.0 μm long. In bovine adrenal cortex the shape of catalase-positive particles is rather spherical (diameter 0.1 to 0.3 μm). Particles of both organs lack crystalline or dense cores.Biochemical examination of cell fractions prepared from tissue homogenates by differential centrifugation revealed the presence of two typical peroxisomal oxidases, viz. α-hydroxy acid and -amino acid oxidase, with maximal relative specific activities in the ‘microsomal’ fraction (preputial gland) and in the ‘lysosomal’ fraction (adrenal cortex), respectively. Urate oxidase is absent in both tissues.The concomitant occurrence of catalase and hydrogen peroxide producing oxidases in the particles described characterizes them as true peroxisomal systems (‘microperoxisomes’).     

Ultrastructural localization of D-amino acid oxidase in microperoxisomes of the rat nervous system.

A recently developed procedure for the localization of D-amino acid oxidase (D-AAO) has been used to investigate the distribution of this enzyme in rat nervous tissue. Initial studies were carried out on kidney to validate the methods. The cytochemically demonstrable enzyme in kidney is inhibited by kojic acid, a known competitive D-AAO inhibitor. Omission of the catalse inhibitor, aminotriazole, from the cytochemical medium produces a marked diminution of D-AAO reaction product in kidney peroxisomes. This would be expected if catalase and D-AAO are present in the same particles. In brain, kojic acid-inhibitable D-AAO is demonstrable in numerous bodies within astrocytes especially in the cerebellum, a brain region known from biochemistry to contain particularly high levels of the oxidase. In preparations incubated for catalase, far fewer positive bodies are seen in the cerebellum. Moreover, omission of aminotriazole has little evident effect on the D-AAO reaction. Thus, the oxidase-containing cerebellar bodies may be relatively poor in catalse. In contrast, several nervous system cell types that contain relatively numerous catalase-positive bodies, contain none with detectable D-AAO. Such heterogeneity of peroxisome enzyme content is in accord with reports from biochemical studies of brain.     

Activity of peroxisomal enzymes and intracellular distribution of catalase in Zellweger syndrome

The activity of peroxisomal enzymes was studied in human liver and cultured human skin fibroblasts in relation to the finding (Goldfischer, S. et al. (1973) Science 182, 62-64) that morphologically distinct peroxisomes are not detectable in patients with the cerebro-hepato-renal (Zellweger) syndrome. In homogenates of liver from the patients, dihydroxyacetone phosphate acyltransferase, a membrane-bound peroxisomal enzyme, is deficient (Schutgens, R.B.H., et al. (1984) Biochem. Biophys. Res. Commun. 120, 179-184). In contrast, there is no deficiency of the soluble peroxisomal matrix enzymes catalase, L-alpha-hydroxyacid oxidase and E-aminoacid oxidase. Catalase is also not deficient in homogenates of cultured skin fibroblasts from the patients. The results of digitonin titration experiments showed that in control fibroblasts at least 70% of the catalase activity is present in subcellular particles distinct from mitochondria or lysosomes. In contrast, all of the catalase activity in fibroblasts from Zellweger patients is found in the same compartment as the cytosolic marker enzyme lactate dehydrogenase.     

Peroxisomes in human colon carcinomas. A cytochemical and biochemical study.

The presence of peroxisomes and their enzymic content were investigated and compared in healthy and neoplastic human colon epithelial cells using cytochemical studies at the ultrastructural level as well as biochemical analyses. Catalase-positive organelles were found to be more numerous in normal than in colonic neoplastic cells. Biochemical assays revealed that no D-aminoacid oxidase or L-alpha-hydroxyacid oxidase activity was detected in normal or tumor tissues. The specific activities of catalase, fatty-acyl CoA oxidase and enoyl-CoA hydratase/3 hydroxyacyl-CoA dehydrogenase (the so-called peroxisomal bifunctional enzyme of the beta-oxidation system) were found to be diminished in carcinoma cells compared with the control tissue. The fall in catalase activity correlated well with tumor stage according to Dukes, suggesting that this peroxisomal enzyme could be used as a potential prognostic marker.     

Cytochemical demonstration of catalasepositive particles (peroxisomes?) in fibroblasts

Summary The connective tissue of the mucosa of the respiratory tract, of the gastric mucosa and of the mucosa of the tongue was investigated in mice. The tissue was fixed in glutaraldehyde and incubated in an alkaline DAB-medium to demonstrate the peroxidatic activity of catalase. In fibroblasts and fibrocytes, as well as in lymphoid cells, membrane bounded particles from 0.10 to 0.25 m in diameter were found, whose matrices were intensely stained by the histochemical reaction. The reaction is inhibited by the addition of 2×10^–2 M 3-amino-1,2,4-triazole. In connective tissue cells of specimens, which were not reacted to demonstrate catalase activity, these organelles show a granular matrix of moderate electron density. They lack a crystalline core. The possibility that these catalase-positive particles (CPs) represent peroxisomes is discussed.This investigation was kindly supported by a grant of Hochschuljubiläumsstiftung der Stadt Wien.     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

A novel analytical method to evaluate directly catalase activity of microorganisms and mammalian cells by ESR oximetry

Abstract

Electron spin resonance (ESR) oximetry technique was applied for analysis of catalase activity in the present study. Catalase activity was evaluated by measuring oxygen from the reaction between hydrogen peroxide (H₂O₂) and catalase-positive cells. It was demonstrated that the ESR spectra of spin-label probes, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO) and 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy (4-maleimido-TEMPO) in the presence of H₂O₂ were broadened with the concentrations of catalase. It was possible to make a calibration curve for catalase activity by peak widths of the spectra of each spin-label probe, which are broadened dependently on catalase concentrations. The broadened ESR spectra were also observed when the catalase-positive micro-organisms or the mammalian cells originally from circulating monocytes/macrophages were mixed with TEMPOL and H₂O₂. Meanwhile, catalase-negative micro-organisms caused no broadening change of ESR spectra. The present study indicates that it is possible to evaluate directly the catalase activity of various micro-organisms and mammalian cells by using an ESR oximetry technique.     

A Survey of Plants for Leaf Peroxisomes

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.     

Studies on peroxisomes III. Further studies on the intraparticulate localization of peroxisomal components in the liver of the rat

The peroxisome-rich fraction prepared from rat liver homogenate was treated by various procedures and the behavior of the peroxisomal core on sucrose density gradient centrifugation was investigated.Peroxisomes were destroyed by various treatments, such as pH 9.0, VirTis blender, sonication and deoxycholate, resulting in the solubilization of catalase from the particles. Urate oxidase was not solubilized at all such treatments. Although D-amino acid oxidase was solubilized by treatments with deoxycholate and VirTis blender, this enzyme was found to be resistant to solubilization by treatment with pH 9.0 or sonication, in contrast to catalase.When the peroxisomal core was investigated, using urate oxidase activity as a marker, its density proved to be changed when submitted to various treatments. These results indicated that the peroxisomes consist of four compartments: a catalase-containing compartment (matrix), a urate oxidase containing compartment (core), a D-amino acid oxidase containing compartment and a low density compartment which is proposed for the first time in the present paper. Furthermore, it was also found that the last two compartments seem to be bound to the core, though the binding might be weak.     

Characterization of the oxidase activity in mammalian catalase

Catalase is a highly conserved heme-containing antioxidant enzyme known for its ability to degrade hydrogen peroxide into water and oxygen. In low concentrations of hydrogen peroxide, the enzyme also exhibits peroxidase activity. We report that mammalian catalase also possesses oxidase activity. This activity, which is detected in purified catalases, cell lysates, and intact cells, requires oxygen and utilizes electron donor substrates in the absence of hydrogen peroxide or any added cofactors. Using purified bovine catalase and 10-acetyl-3,7-dihydroxyphenoxazine as the substrate, the oxidase activity was found to be temperature-dependent and displays a pH optimum of 7-9. The Km for the substrate is 2.4 x 10(-4) m, and Vmax is 4.7 x 10(-5) m/s. Endogenous substrates, including the tryptophan precursor indole, the neurotransmitter precursor beta-phenylethylamine, and a variety of peroxidase and laccase substrates, as well as carcinogenic benzidines, were found to be oxidized by catalase or to inhibit this activity. Several dietary plant micronutrients that inhibit carcinogenesis, including indole-3-carbinol, indole-3-carboxaldehyde, ferulic acid, vanillic acid, and epigallocatechin-3-gallate, were effective inhibitors of the activity of catalase oxidase. Difference spectroscopy revealed that catalase oxidase/substrate interactions involve the heme-iron; the resulting spectra show time-dependent decreases in the ferric heme of the enzyme with corresponding increases in the formation of an oxyferryl intermediate, potentially reflecting a compound II-like intermediate. These data suggest a mechanism of oxidase activity involving the formation of an oxygen-bound, substrate-facilitated reductive intermediate. Our results describe a novel function for catalase potentially important in metabolism of endogenous substrates and in the action of carcinogens and chemopreventative agents.     

Purification and immuno-electron microscopical characterization of crystalline inclusions from plant peroxisomes

Summary This paper describes the first purification method for crystalline inclusions (cores) from plant peroxisomes and an ultrastructural characterization of these isolated cores. 5-day-old sunflower (Helianthus annuus L.) cotyledon fractions which were highly enriched in cores showed negligible activity of the matrix enzyme glycolate oxidase but high catalase activity. As proven by electron microscopy, crystalline particles were surrounded neither by matrix material nor by membranes. Their geometrical outlines and ultrastructure were identical to those of cores in tissue sections, as was their reactivity with three different polyclonal catalase antibodies in the immunogold technique. Three-dimensional reconstruction, based on the geometrical outlines and ultrastructure of sectioned isolated cores from sunflower, suggested that they were quadrangular blocks. Ultrastructural analysis revealed an even periodic arrangement of repeating units which are probably cubes with 20 nm long edges. Isolated peroxisomal cores from potato (Solanum tuberosum L.) tubers had outlines which suggested that they were even rhomboidal prisms. They showed a granular ultrastructure without any repeating units and contained catalase, demonstrated by immunogold labelling and enzyme activity measurement. The results presented here suggested the hypothesis that the structural elements in plant peroxisomal cores are made of enzymatically active catalase, although the substructure may vary from species to species.Abbreviations ACOx acyl-CoA oxidase - BSA bovine serum albumin - EDTA ethylenediamine-tetraacetate - GDH glutamate dehydrogenase - GOx glycolate oxidase - KPB potassium phosphate buffer     

Structure, composition, physical properties, and turnover of proliferated peroxisomes. A study of the trophic effects of Su-13437 on rat liver

Peroxisome proliferation has been induced with 2-methyl-2-(p-[1,2,3,4-tetrahydro-1-naphthyl]-phenoxy)-propionic acid (Su-13437). DNA, protein, cytochrome oxidase, glucose-6-phosphatase, and acid phosphatase concentrations remain almost constant. Peroxisomal enzyme activities change to approximately 165%, 50%, 30%, and 0% of the controls for catalase, urate oxidase, L-alpha-hydroxy acid oxidase, and D-amino acid oxidase, respectively. For catalase the change results from a decrease in particle-bound activity and a fivefold increase in soluble activity. The average diameter of peroxisome sections is 0.58 +/- 0.15 mum in controls and 0.73 +/- 0.25 mum after treatment. Therefore, the measured peroxisomal enzymes are highly diluted in proliferated particles. After tissue fractionation, approximately one-half of the normal peroxisomes and all proliferated peroxisomes show matric extraction with ghost formation, but no change in size. In homogenates submitted to mechanical stress, proliferated peroxisomes do not reveal increased fragility; unexpectedly, Su-13437 stabilizes lysosomes. Our results suggest that matrix extraction and increased soluble enzyme activities result from transmembrane passage of peroxisomal proteins. The changes in concentration of peroxisomal oxidases and soluble catalase after Su-13437 allow the calculation of their half-lives. These are the same as those found for total catalase, in normal and treated rats, after allyl isopropyl acetamide: about 1.3 days, a result compatible with peroxisome degradation by autophagy. A sequential increase in liver RNA concentration, [14C]leucine incorporation into DOC-soluble proteins and into immunoprecipitable catalase, and an increase in liver size and peroxisomal volume per gram liver, characterize the trophic effect of the drug used. In males, Su-13437 is more active than CPIB, another peroxisome proliferation-inducing drug; in females, only Su-13437 is active.     

Cytochemical localization of catalase and several hydrogen peroxide-producing oxidases in the nucleoids and matrix of rat liver peroxisomes

Synopsis The distribution of catalase, amino acid oxidase, -hydroxy acid oxidase, urate oxidase and alcohol oxidase was studied cytochemically in rat hepatocytes. The presence of catalase was demonstrated with the conventional diaminobenzidine technique. Oxidase activities were visualized with methods based on the enzymatic or chemical trapping of the hydrogen peroxide produced by these enzymes during aerobic incubations.All enzymes investigated were found to be present in peroxisomes. Catalase activity was found in the peroxisomal matrix, but also associated with the nucleoid. After staining for oxidase activities the stain deposits occurred invariably in the peroxisomal matrix as well as in the nucleoids. In all experiments the activity of both catalase and the oxidases was confined to the peroxisomes. The presence of a hydrogen peroxide-producing alcohol oxidase was demonstrated for the first time in peroxisomes in liver cells.The results imply that the enzyme activity of the nucleoids of rat liver peroxisomes is not exclusively due to urate oxidase. The nucleoids obviously contain a variety of other enzymes that may be more or less loosely associated with the insoluble components of these structures.     

Biochemical, electrophoretic and immunological characterization of peroxisomal enzymes in three soybean organs

Biochemical, electrophoretic and immunological studies were made among peroxisomal enzymes in three organs of soybean [Glycine max (L.) Merr. cv. Centennial] to compare the enzyme distribution and characteristics of specialized peroxisomes in one species. Leaves, nodules and etiolated cotyledons were compared with regard to several enzymes localized solely in their peroxisomes: catalase (EC 1.11.1.6), malate synthase (EC 4.1.3.2), glycolate oxidase (EC 1.1.3.1), and urate oxidase (EC 1.7.3.3). Catalase activity was found in all tissue extracts. Electrophoresis on native polyacrylamide gels indicated that leaf catalase migrated more anodally than nodule or cotyledon catalase as shown by both activity staining and Western blotting. Malate synthase activity and immunologically detectable protein were present only in the cotyledon extracts. Western blots of denaturing (lithium dodecyl sulfate) gels probed with anti-cotton malate synthase antiserum, reveal a single subunit of 63 kDa in both cotton and soybean cotyledons. Glycolic acid oxidase activity was present in all three organs, but ca 20-fold lower (per mg protein) in both nodule and cotyledon extracts compared to leaf extracts. Electrophoresis followed by activity staining on native gels indicated one enzyme form with the same mobility in nodule, cotyledon and leaf preparations. Urate oxidase activity was found in nodule extracts only. Native gel electrophoresis showed a single band of activity. Novel electrophoretic systems had to be developed to resolve the urate oxidase and glycolate oxidase activities; both of these enzymes moved cathodally in the gel system employed while most other proteins moved anodally. This multifaceted study of enzymes located within three specialized types of peroxisomes in a single species has not been undertaken previously, and the results indicate that previous comparisons between the enzyme content of specialized peroxisomes from different organisms are mostly consistent with that for a single species, soybean.     

Peroxisomes and peroxisomal functions in muscle. Studies with muscle cells from controls and a patient with the cerebro-hepato-renal (Zellweger) syndrome

In the present study we investigated peroxisomal functions in cultured human muscle cells from control subjects and from a patient with the Zellweger syndrome, a genetic disease characterized by the absence of morphologically distinguishable peroxisomes in liver and kidney. In homogenates of cultured muscle cells from control subjects, catalase is contained within subcellular particles, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity is present and palmitoyl-CoA can be oxidized by a peroxisomal beta-oxidative pathway; these findings are indicative of the presence of peroxisomes in the cells. In homogenates of cultured muscle cells from the patient with the Zellweger syndrome, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity was deficient, peroxisomal beta-oxidation of palmitoyl-CoA was impaired and catalase was not particle-bound. These findings indicate that functional peroxisomes are absent in muscle from patients with the Zellweger syndrome. We conclude that cultured human muscle cells can be used as a model system to study peroxisomal functions in muscle and the consequences for this tissue of a generalized dysfunction of peroxisomes.     

On the compartmentalization of catalase,fatty acyl-CoA oxidase and urate oxidase in mammalian livers,and the influence of clofibrate treatment on this microlocalization

The compartmentalization of catalase, fatty acyl-CoA oxidase and urate oxidase was examined in the livers of mice, rats and guinea pigs, using the technique of digitonin extraction in order to avoid the trauma associated with centrifugation procedures. The results are interpreted as indicating that an appreciable proportion of catalase activity occurs in the cytoplasmic compartment of these cells. Following treatment of the animals with clofibrate, the specific activity in both peroxisomal and cytoplasmic compartments was increased, with a higher proportion of cytoplasmic catalase being evident in mice. The results for catalase were compared with those for fatty acyl-CoA oxidase and urate oxidase both of which were indicated as showing a closer association with the peroxisomal compartment than was the case for catalase. These data have been discussed in relation to their significance on present understanding of peroxisomal structure and function.     

Degradation of peroxisomal catalase and urate oxidase of rat liver.

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5--9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation. After [3H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37 degrees C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process. From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.     

Studies on bone enzymes. Distribution of acid hydrolases, alkaline phenylphosphatase, cytochrome oxidase and catalase in subcellular fraction of bone tissue homogenates

1. When bone homogenates were fractionated according to the scheme developed for liver by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955), all the enzymes assayed except cytochrome oxidase were found to occur partly in soluble and partly in particulate fractions. Among the particle-bound enzymes, the highest specific activity was found in the heavy-mitochondrial fraction for cytochrome oxidase, in the microsomal fraction for alkaline phenylphosphatase and in the light-mitochondrial fraction for eight acid hydrolases and for catalase. 2. Combined heavy-mitochondrial and light-mitochondrial fractions were subfractionated by isopycnic centrifugation in density gradients of sucrose or glycogen. In the various systems tried, cytochrome oxidase showed a relatively narrow distribution range with a sharp peak; the acid hydrolases and catalase showed flat and irregular distribution patterns, differing slightly in shape from one enzyme to the other. However, it was not possible to achieve a marked separation between the various enzymes under study. 3. It is concluded from these results that the acid hydrolases belong to special cytoplasmic particles, probably lysosomes, and that these particles are physically and enzymically heterogeneous. Catalase appears to be non-mitochondrial and could also belong to the lysosomes; but the possibility of an association with another type of particle must be kept in mind in view of what is known of liver catalase. Alkaline phenylphosphatase is largely attached to microsomal elements.     

Degradation of peroxisomal catalase and urate oxidase of rat liver

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5–9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation.After [³H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37°C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process.From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.