Quantitation of catalase activity by microspectrophotometry after diaminobenzidine staining

Summary The absorbance of the reaction product of catalase staining with diaminobenzidine is linearly proportional to enzyme activity. This is shown in semithin Epon sections of model systems containing serum albumin and catalase from bovine or guinea pig liver. Absorbance measurements were also performed on semithin sections of guinea pig liver, and from these, the activity of cytoplasmic (extraperoxisomal) catalase has been derived.I.W.O.N.L. bursaal 1977–1979     

Catalase in guinea pig hepatocytes is localized in cytoplasm, nuclear matrix and peroxisomes

We have compared the intracellular localization of catalase and another peroxisomal marker enzyme, alpha-hydroxy acid oxidase (HAOX), in the livers of guinea pig and rat using immunoelectron microscopy and subcellular fractionation combined with immunoblotting and enzyme activity determination. Antibodies against both enzymes were raised in rabbits and their specificities established by immunoblotting. By immunoelectron microscopy, gold particles representing antigenic sites for catalase were found in guinea pig hepatocytes not only in peroxisomes but also in the cytoplasm and the nuclear matrix. In rat liver, however, catalase was localized exclusively in peroxisomes with no cytoplasmic labeling. Moreover, in both species HAOX was found only in peroxisomes. Subcellular fractionation revealed that purified peroxisomes from both species contained comparable levels of each, catalase and HAOX activities. The total catalase activity, however, was substantially higher in guinea pig and most of this excess catalase was in the cytosolic fraction with some activity also in nuclei. In rat liver, 30 to 40% of both enzymes and in guinea pig liver 30% of HAOX were recovered in the supernatant fraction implying that the fragility of peroxisomes in both species is quite comparable. These observations establish the occurrence of extraperoxisomal catalase in guinea pig liver. The catalase in the cytoplasm and nucleus of liver parenchymal cells is most probably involved in scavenging of H2O2, protecting the cell against toxic and mutagenic effects of this noxious agent.     

On the topology of the catalase biosynthesis and -degradation in the guinea pig liver. A cytochemical study

The biosynthesis, transport and degradation of catalase have been studied in the guinea pig liver parenchymal cell using 2-allyl-2-isopropylacetamide (AIA) as an inhibitor of de novo formation of catalase. Total catalase activity was assayed biochemically; cytoplasmic catalase was measured microspectrophotometrically after quantitative diaminobenzidine staining of the liver. By morphometry, number and size of peroxisomes in catalase stained sections were determined. From our data we conclude that (1) the final step in the catalase formation takes place inside peroxisomes, (2) catalase is transported from the peroxisomes into the cytoplasm, (3) in the cytoplasm catalase is degraded. These conclusions in part confirm the topological model on the intracellular catalase biosynthesis pathway of Lazarow and de Duve (1973) except for the presence of cytoplasmic catalase which is released from the peroxisomes as proposed earlier by Jones and Masters (1975).     

Enhanced heme oxygenase activity increases the antioxidant defense capacity of guinea pig liver upon acute cobalt chloride loading: comparison with rat liver

Changes in the activity of so-called oxidative stress defensive enzymes, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and heme oxygenase, as well as changes in lipid peroxidation and reduced glutathione levels, were measured in guinea pig and rat liver after acute cobalt loading. Cobalt chloride administration produced a much higher degree of lipid peroxidation in guinea pig than in rat liver compared with the control animals. The intrahepatic reduced glutathione content in control guinea pig was higher than that in rat, but was equally decreased in both species after cobalt administration. The enzymatic scavengers of free radicals, superoxide dismutase, catalase and glutathione peroxidase, were significantly decreased in rat liver after acute cobalt loading, and as a compensatory reaction, the heme oxygenase activity was increased (seven-fold). In guinea pig liver, only superoxide dismutase activity was depleted in response to cobalt-induced oxidative stress, while catalase and glutathione peroxidase were highly activated and the heme oxygenase activity was dramatically increased (13-fold). It is assumed that enhanced heme oxygenase activity may have important antioxidant significance by increasing the liver oxidative-stress defense capacity.     

On the topology of the catalase biosynthesis and-degradation in the guinea pig liver

Summary The biosynthesis, transport and degradation of catalase have been studied in the guinea pig liver parenchymal cell using 2-allyl-2-isopropylacetamide (AIA) as an inhibitor of de novo formation of catalase. Total catalase activity was assayed biochemically; cytoplasmic catalase was measured microspectrophotometrically after quantitative diaminobenzidine staining of the liver. By morphometry, number and size of peroxisomes in catalase stained sections were determined. From our data we conclude that (1) the final step in the catalase formation takes place inside peroxisomes, (2) catalase is transported from the peroxisomes into the cytoplasm, (3) in the cytoplasm catalase is degraded. These conclusions in part confirm the topological model on the intracellular catalase biosynthesis pathway of Lazarow and de Duve (1973) except for the presence of cytoplasmic catalase which is released from the peroxisomes as proposed earlier by Jones and Masters (1975).     

Investigation of peroxisomal lipid beta-oxidation enzymes in guinea pig liver peroxisomes by immunoblotting and immunocytochemistry.

We investigated the immunoreactivity of the peroxisomal lipid beta-oxidation enzymes acyl-CoA oxidase, trifunctional protein, and thiolase in guinea pig liver and compared it with that of homologous proteins in rat, using immunoblotting of highly purified peroxisomal fractions and monospecific antibodies to rat proteins. In addition, the immunocytochemical localization of beta-oxidation enzymes in guinea pig liver was compared with that of catalase. All antibodies showed crossreactivity between the two species, indicating that these peroxisomal proteins have been well conserved, although all exhibited some differences with respect to molecular size and, in the case of acyl-CoA oxidase, in frequency of the immunoreactive bands. In the latter case, a distinct second band in the 70 KD range was observed in guinea pig, in addition to the regular band due to subunit A present in rat liver. This novel band could be due either to trihydroxycoprostanoyl-CoA oxidase or to the non-inducible branched chain fatty acid oxidase described recently. All three beta-oxidation enzymes were immunolocalized by light and electron microscopy to the matrix of peroxisomes, in contrast to catalase, which is also found in the cytoplasm and the nucleus of hepatocytes in guinea pig liver.     

Hypoxia and cold: influence on cellular oxygen consuming systems in guinea pig liver

Traditionally any biochemical changes found in animals exposed to high altitude have been interpreted solely in relation to hypobaric hypoxia. The present work has been carried out to study the influence of cold and hypoxia in guinea pig native to high altitude.The three major oxygen consuming systems of the liver were measured using cytochrome oxidase as a mitochondrial marker, catalase as a peroxisomal marker, and the o-demethylation of p-nitroanisole and the hydroxilation of hexobarbital as markers for microsomal activity. Serum levels of thyroid hormones (T₃ and T₄) were also determined.Evidence is presented showing that cold produces a dramatic increase of liver catalase and cytochrome oxidase activities, and of serum T₃ and T₄.Interestingly, the increase in thyroid hormones did not precede the increase of the two enzyme activities in the liver of guinea pig exposed to 4°C.On the other hand, it was found that hypoxia appears to have no significant influence upon any of the three major oxygen consuming systems of the liver.     

Palmityl-CoA oxidase: Detection in several guinea pig tissues and peroxisomal localisation in mucosa of small intestine

Using a sensitive assay palmityl-CoA oxidase activity was detected in several guinea pig tissues including liver, kidney, small intestine, lung, muscle, spleen and heart. Subcellular fractionation using sucrose and metrizamide gradients demonstrated that in small intestinal mucosa palmityl-CoA oxidase is localised in the same particles as catalase. These observations are discussed in the context of a role for peroxisomal fatty acid oxidation in energy balance and thermogenesis.     

Demonstration of apolipoprotein CII in guinea pigs. Functional characteristics, cDNA sequence, and tissue expression

In contrast to plasma from other mammals, guinea pig plasma does not stimulate the activity of lipoprotein lipases in vitro. This had led previously to the conclusion that guinea pigs lack an analogue to apolipoprotein CII (apoCII). By adsorption of lipid-binding proteins to lipid droplets, thereby separating them from other plasma components, we could demonstrate apoCII-like activity in guinea pig plasma. On electrophoresis, the CII-like activity co-migrated with one isoform of guinea pig apolipoprotein CIII, identified by amino-terminal amino acid sequence determination (40 residues). By isoelectric focusing in a narrow pH gradient, the activating protein was separated sufficiently from the dominating apoCIII isoform to allow sequence determination of 8 residues from the amino terminus. Six of these were identical to corresponding residues in apoCII from dog and monkey. With the aid of a human apoCII cDNA probe we identified one cross-hybridizing mRNA species (approximately 600 nucleotides) on Northern blots of guinea pig liver. Three positive clones were isolated from a guinea pig liver cDNA library using the same cDNA probe. The nucleotide sequence showed extensive similarities to the previously known human, monkey, and canine sequences, but the signal peptide was 3 amino acid residues longer in the guinea pig protein, and there was a deletion of 4 residues in the putative lipid binding domain. Northern blot analyses indicated that guinea pig apoCII is mainly expressed in the liver with little or no contribution from the intestine.     

GTP hydrolysis by guinea pig liver transglutaminase

Homogeneous guinea pig liver transglutaminase was purified from a commercially available enzyme preparation by affinity chromatography on GTP-agarose. The purified transglutaminase exhibited a single band of apparent Mr = 80,000 on sodium dodecyl sulfate polyacrylamide gel and Western blotting and had enzyme activity of both transglutaminase and GTPase. The guinea pig liver transglutaminase has an apparent Km value of 4.4 microM for GTPase activity. GTPase activity was inhibited by guanine nucleotides in order GTP-gamma-S greater than GDP, but not by GMP. These results demonstrate that purified guinea pig liver transglutaminase catalyzes GTP hydrolysis.     

An immunological investigation of catalase in three mammalian species

An immunological comparison of catalase has been conducted in the mouse, rat and guinea pig. In each of these species, enzyme activity from the peroxisomal compartment was indicated as immunologically distinct from the cytosolic activity, with catalase in the small granule compartment displaying intermediate characteristics. When related to the established data on the biogenetic and degradative changes of catalase in the mouse, the present results support the proposal that peroxisomal alterations, epigenetic modifications and degradative persistence are general characteristics of catalase biology in mammalian species.     

Immunocytochemical demonstration of LH-RH, somatostatin, and ACTH-like peptide in osmium-postfixed, resin-embedded median eminence

In guinea pig median eminences that were fixed with a glutaraldehyde paraformaldehyde mixtures, postfixed with osmium tetroxide, and embedded in araldite, immunocytochemical stainings for luteinizing hormone-releasing hormone (LH-RH), somatostatin, and alpha 17-39 ACTH have been obtained on semithin sections. These positive reactions were obtained after araldite was removed with sodium methoxide and after treatment with hydrogen peroxide. These data suggest that osmium tetraoxide postfixation can be used for the detection of low molecular weight peptides such as LH-RH, somatostatin, and ACTH-like-peptides not only in the median eminence but also in the hypothalamus.     

Studies on the effect of soluble lymphocyte products (lymphokines) on macrophage physiology. I. Early changes in enzyme activity and permeability.

Various cytochemical techniques have been used to quantitate the rapid effect of a partially purified, soluble product from lymphocytes (lymphokine) on normal guinea pig macrophages in vitro. Early changes in the utilisation of hydrogen liberated from the hexose monophosphate shunt and on cellular permeability were observed. The ability of the lymphokine to alter hydrogen utilisation was also seen in experiments on cryostat sections of guinea pig liver, suggesting that the cytochemical effects were not predetermined by changes at the membrane level. It is suggested that lymphokine-induced changes within the cell may reduce some biosynthetic activity affecting the cell membrane and this may in part reflect the decreased migrating ability of the cells. Increases in NADPH oxidation after lymphokine contact are discussed in relation to the bactericidal capacity of the cells.     

Characterization of insulinase from mammalian and non-mammalian livers

The Michaelis constants (Km's) and maximum reaction velocities (Vmax's) for the degradation of beef insulin by livers from frogs, guinea pigs, rats, a rabbit, a dog and a pig were determined. The Km's for mammalian livers appear to be species-dependent and range from 0.25 microM to 0.65 microM. The Km for frog liver was somewhat lower, averaging 0.13 microM. The Km is independent of animal age, but the enzyme concentrations (Vmax) were greatly reduced in the fetal guinea pig and 3 day rat compared to the adult livers. There appears to be no relation between Km and the chemical dissimilarity between beef insulin and endogenous insulin of the species, since guinea pig liver insulinase had a Km (0.50 microM) intermediate between dog (0.47 microM) and pig (0.65 microM) liver insulinase although guinea pig insulin has a markedly different amino acid sequence and biologic activity.     

Chromatofocusing of mammalian estrone sulfate sulfohydrolase activity

Estrone sulfate sulfohydrolase (estrogen sulfatase) activity was solubilized by treatment with Triton X-100 from 105,000 g pellets of guinea pig uterus, testis and brain, as well as from rat liver and human placenta. The solubilized forms were subjected to chromatofocusing in the fast protein liquid chromatography (FPLC) system and on conventional columns packed in our laboratory. The guinea pig tissue pattern was complex. Uterus showed peaks of activity with apparent pI's of 9.11 and 7.6; testis contained 3 peaks with pI's of 9.18, 8.7 and 7.5; brain possessed peaks with pI's of 9.28 and 8.6. In each case the major activity peak was that with pI greater than 9. Rat liver activity chromatofocused as a single peak of apparent pI = 6.87 and the human placental enzyme also showed a single, though broad, peak, of pI = 6.57. This suggests not only that the guinea pig enzyme(s) differs markedly from those of rat liver and human placenta, but that there may be qualitative differences between the forms in the three guinea pig tissues. Chromatofocusing behaviour was not independent of the specific exchange resins and ampholytes utilized. The recovered enzyme activity was fairly stable and it seems that chromatofocusing could be a useful step in purification of the guinea pig enzyme(s), particularly the main form possessing a pI greater than 9.     

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase

Summary The feasibility of the application of a television-based image analyzer, the Texture Analysis System (TAS, Leitz Wetzlar, FRG) in conjunction with a light microscope for morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained with the alkaline-DAB method for localization of catalase and semi-thin (0.25 and 1 m) sections of plastic-embedded material were examined under an oil immersion objective. The TAS detected the peroxisomal profiles selectively and determined their morphometric parameters automatically. The same parameters were obtained also by morphometric analysis of electron micrographs from the same material. The volume density of peroxisomes determined by TAS in semithin sections of normal liver, after correction for section thickness, is quite close to the corresponding value obtained by morphometry of electron micrographs. The difference is approximately 20%. In animals treated with the hypolipidemic drug bezafibrate, which causes proliferation of peroxisomes, TAS detected readily the increase in volume density of peroxisomes in semithin sections. In comparison with electron microscopy, however, the light-microscopic approach seems to underestimate the proliferation. The lower resolution of the light microscope and overlapping of neighbouring particles in relatively thick sections used for lightmicroscopic analysis may account for the differences.The present study has demonstrated the usefulness of automatic image analysis in conjunction with selective cytochemical staining of peroxisomes for morphometry of this organelle in rat liver. The light-microscopic approach is not only faster but is also extremely economical by obviating the use of an electron microscope.     

On the turnover and proteolysis of catalase in tissues of the guinea pig and acatalasemic mice.

Turnover characteristics (half-lives and rate constants for synthesis and degradation) have been determined for the catalases of guinea pig and three different strains of mice by means of the kinetics of return of enzyme activity after inhibition with 3-amino-1,2,4-triazole. The catalase of hypocatalasemic mice (strain C_sD) did not display an appreciably different half-life to that of the wild-type mice, but catalase in the tissues of acatalasemic mice (strain C_sB) exhibited a half-life which was only half that of the wild type, while the half-life of guinea pig catalase was more than twice that of wild-type mice. Significant differences were also noticed in regard to the in vitro susceptibility of the catalases of these animals to protease inactivation. Large-granule (lysosomal, mitochondrial and peroxisomal) extracts proved far more susceptible to protease inactivation than cytosol extracts, and marked changes in the heteromorph pattern of mouse liver cytosol catalase were observed to accompany limited proteolysis. These results support the conclusion that the in vitro susceptibility of proteases may be an important determining factor in the rate of degradation of an enzyme in vivo.     

Oxidative metabolism of chicken polymorphonuclear leucocytes during phagocytosis

Summary The oxidative response to phagocytosis by chicken polymorphonuclear leucocytes was investigated as compared to guinea pig polymorphonuclear leucocytes.The polymorphs from both species respond to phagocytosis with an increased oxygen consumption, an increased generation of O₂ ^– and H₂O₂, and an increased oxidation of glucose through the hexose monophosphate shunt. The rate of oxygen consumption, and generation of O₂ ^– and H₂O₂ by phagocytosing chicken polymorphonuclear leucocytes is considerably lower than with phagocytosing guinea pig polymorphonuclear leucocytes. By contrast, the extent of hexose monophosphate shunt stimulation in chicken polymorphs is comparable to that of guinea pig polymorphs. Evidence is presented suggesting that H₂O₂ is preferentially degraded in chicken cells through the glutathione cycle, whereas catalase and myeloperoxidase are the two main H₂O₂ degrading enzymes in guinea pig cells.The 20,000 g fraction of the postnuclear supernatant of chicken polymorphs contains a cyanide-insensitive NADPH oxidizing activity which is stimulated during phagocytosis. Similar properties for the NADPH oxidizing activity of guinea pig polymorphs have been previously reported.It is concluded that the metabolic burst of phagocytosing chicken polymorphonuclear leucocytes is qualitatively similar to that of guinea pig polymorphonuclear leucocytes, but the latter cells are more active in all the biochemical parameters that have been measured. The difference in the H₂O₂ degradation pathways between the two species is accounted for by the lack of myeloperoxidase and catalase in chicken polymorphs.