Rat Liver Catalase Research Articles

Containing catalase in rat liver

Containing catalase in rat liver

Detection of catalase in rat heart mitochondria.

The presence of heme-containing catalase in rat heart mitochondria (20 +/- 5 units/mg) was demonstrated by biochemical and immunocytochemical analysis. Intact rat heart mitochondria efficiently consumed exogenously added H2O2. The rate of H2O2 consumption was not influenced by succinate, glutamate/malate, or N-ethylmaleimide but was significantly inhibited by cyanide. Hydrogen peroxide decomposition by mitochondria yielded molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism. Mitochondrial fractionation studies and quantitative electron microscopic immunocytochemistry revealed that most catalase was matrix-associated. Electrophoretic analysis and Western blotting of the mitochondrial matrix fraction indicated the presence of a protein with similar electrophoretic mobility to bovine and rat liver catalase and immunoreactive to anti-catalase antibody. Myocardial tissue has a lower catalase-specific activity and a greater mitochondrial H2O2 production/g of tissue than most organs. Thus catalase, representing 0.025% of heart mitochondrial protein, is important for detoxifying mitochondrial derived H2O2 and represents a key antioxidant defense mechanism for myocardial tissue.

Analysis of RNA for transcripts for catalase and SP71 in rat hearts after in vivo hyperthermia

Hyperthermic stress induces synthesis of the major inducible (heat) stress protein (SP71) in all rat tissues. In addition, there is an increase in catalase activity in hearts at 24 and 48 h after the induction of the heat shock response. To more precisely define some of the molecular aspects of the induction of the heat shock response in hearts, we examined mRNA levels for the catalase, SP71 and HSP27. RNA was isolated from control hearts and at various time periods (0-24 h) of recovery after brief hyperthermic treatment and was analyzed by Northern blot analysis using as probes cDNA sequences for rat liver catalase, human HSP70 (inducible), and human HSP27. There was no detectable change in mRNA for catalase after heat shock or during recovery. Hyperthermic stress has no apparent effect on the regulation of transcription of mRNA coding for catalase, indicating that the increase in catalase activity is either translationally or post-translationally regulated. The human HSP70 cDNA did not hybridize to control heart RNA, but did hybridize to SP71 transcripts at 0, 1.5, and 3 h post heat shock. The mRNA level for SP71 peaked at 1.5 h, was reduced at 3 h, and became almost undetectable at 6 h post heat shock. Similarly, the human HSP27 cDNA did not hybridize to control heart RNA, but did hybridize to transcripts for HSP27 at 0, 1.5, 3, and up to 15 h post heat shock. Maximal signal for HSP27 was at 3 h post heat shock and was sharply reduced at 6 h post heat shock.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and Properties of Recombinant Rat Catalase Produced in Escherichia coli1

Catalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.

Isolation and characterization of the rat catalase-encoding gene

Using cDNA clones coding for rat liver catalase (hydrogen peroxide: hydrogen peroxide oxidoreductase, EC 1.11.1.6), overlapping genomic clones were isolated. By Southern blotting analysis and nucleotide sequencing, the gene was characterized to be about 33 kb in length and to have 13 exons and twelve introns. S1 mapping and primer extension analyses showed the presence of multiple transcription start points ( tsp) located between 66 bp and 105 bp upstream from the translational start codon. Nucleotide sequence immediately upstream from the tsp lacks the TATA box but contains multiple CCAAT boxes and GC-like boxes.

Glutathione peroxidase and catalase in liver, kidney, testis and brain regions of rats following cadmium exposure and subsequent withdrawal.

Intraperitoneal administration of 0.4 mg/kg Cadmium (Cd) daily for 45 days was found to inhibit the activities of glutathione peroxidase and catalase in liver, kidney, testis and various brain regions at different time intervals. The magnitude of inhibition was increased with the period of exposure. Cd produced significant inhibition of glutathione peroxidase at 15 days in liver, kidney and cerebellum only; however, the enzyme activity was found to be decreased in all the tissues, except corpus striatum, at 30 and 45 days of exposure. Hippocampal glutathione peroxidase remained unaltered throughout the experiment. Catalase was found to be inhibited in all the tissues at different time intervals. The withdrawal of Cd treatment for 15 days after 45 days of exposure did not show significant recovery in the activity of both enzymes of different organs, except kidney and testis where partial and full recoveries respectively were observed. Since these two enzymes constitute an important part of cellular defence mechanism against oxidation, their widespread persistent inhibition may be of great significance in view of the recent reports showing the possible involvement of oxidative stress in the mechanism of Cd toxicity.

Characterization of catalase transcripts and their differential expression in maize

In maize, the three unlinked catalase (EC 1.11.1.6) structural genes ( Cat1, Cat2 and Cat3) are differentially expressed temporally, spatially and in response to environmental signals in the developing seedling. In order to understand more fully the molecular mechanisms involved in catalase gene expression, full-length cDNA clones representing the maize Cat1, Cat2 and Cat3 transcripts were isolated and characterized. DNA sequence analysis confirmed that each cDNA encodes a unique catalase protein. Gene-specific probes for the three maize catalase cDNAs were isolated and used to probe blots of poly(A) + RNA isolated from various maize tissues. Cat1 mRNA was found in scutella, milky endosperm of immature kernels, leaves and epicotyls. The Cat2 mRNA was present primarily in post-germinative scutella, with lower levels in leaves and epicotyls. Cat3 mRNA was detected primarily in epicotyls and, to a lesser extent, in leaves and scutella. The gene-specific probes hybridized with maize genomic DNA blots in simple, but unique patterns, indicating that there is one, or a very few copies of each catalase gene. The coding region of the Cat3 cDNA comprised 66% G + C, which led to a strong codon usage bias in this gene. This codon bias was also seen with the Cat2 transcripts, but not with those for Cat1. A high degree of similarity was found between the maize catalase nucleic acid and deduced amino-acid sequences and those of sweet potato and rat liver catalase.

Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA.

A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313-317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.

Rates of H 2O 2 generation from peroxisomal β-oxidation are sufficient to account for fatty acid-stimulated ethanol metabolism in perfused rat liver

Fatty acids generate H 2O 2 via peroxisomal β-oxidation and increase ethanol metabolism markedly in a system that involves catalase-H 2O 2. The present studies were conducted to understand why fatty acid-stimulated ethanol metabolism occurs much faster than rates of H 2O 2 generation reported previously in perfused rat liver. A new method was developed to measure rates of H 2O 2 generation based on the fact that methanol is oxidized only by catalase in rat liver. Rates of H 2O 2 generation were estimated from the time necessary for the steady-state level of catalase-H 2O 2 measured spectrophotometrically (660-640 nm) through a lobe of the liver to return to basal values following the addition of a known quantity of methanol in a closed perfusion system containing 4% bovine serum albumin. Under these conditions, basal rates of H 2O 2 production and rates of 4-methylpyrazole-insensitive ethanol oxidation were in a similar range (10 to 20 μmol/g/hr). Rates of H 2O 2 generation were increased up to 80 μmol/g/hr by addition of laurate, palmitate or oleate (1 mM); half-maximal increases in rates were observed with 0.6 mM oleate. Hexanoate, a short-chain fatty acid, did not stimulate H 2O 2 production or ethanol uptake. In these studies, rates of H 2O 2 generation compared well with rates of fatty acid-stimulated ethanol uptake measured in the presence of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, with all fatty acids studied. It is concluded, therefore, that rates of H 2O 2 generation are sufficient to account for rates of fatty acid-stimulated ethanol metabolism via catalase-H 2O 2. In addition, these data indicate that catalase may contribute significantly to ethanol oxidation under physiological conditions in the presence of fatty acids.

Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver catalase.

We have isolated five cDNA clones for rat liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6). These clones overlapped with each other and covered the entire length of the mRNA, which had been estimated to be 2.4 kilobases long by blot hybridization analysis of electrophoretically fractionated RNA. Nucleotide sequencing was carried out on these five clones and the composite nucleotide sequence of catalase cDNA was determined. The 5' noncoding region contained 83 bases and was followed by 1581 bases of an open reading frame that encoded 527 amino acids. The 3' noncoding region was 831 bases long and contained long repeats of the unit AC. The amino acid sequence deduced from the nucleotide sequence of the cDNAs showed about 90% homology with the reported primary structure of bovine liver catalase. The molecular weight of rat liver catalase was calculated to be 59,758 from the predicted amino acid sequence. The amino acid residues in contact with the heme group are completely identical for bovine liver and rat liver catalases. The amino acid sequence at the COOH terminus was confirmed by the results of carboxypeptidase P treatment of the protein purified from rat liver in the presence of leupeptin. Rat liver catalase has no cleavable signal peptide for translocation of the enzyme into peroxisomes.

Interaction with Deoxycholate of Rat Liver Peroxisomal and Cytosolic Catalase1

Rat liver catalase was found to interact with deoxycholate (DOC). When purified, the peroxisomal catalase was precipitated at pH 6 in the presence of DOC, whereas in the peroxisomal extract (with DOC) it was unsedimentable at pH 6. The membrane fraction in the extract interacted with the catalase instead of DOC, and prevented the precipitation of catalase with DOC at pH 6. The peroxisomal catalase seemed to be easily modified by lysosomal protease during manipulation, and this proteolytic cleavage rendered the molecule able to interact with the membrane. On the other hand, the cytosolic catalase, both in the cytosol fraction and in the purified preparation, sedimented at pH 6 in the presence of DOC. The cytosolic catalase was far more resistant to proteolytic modification than the peroxisomal catalase. The molecule of peroxisomal catalase is assumed to have a site for recognizing the membrane, whereas such a structure may be absent in the cytosolic catalase or may not be easily exposed by proteolytic cleavage.

Molecular cloning of cDNA for rat liver catalase

For the studies on the induction of peroxisomal enzymes by hypolipidemic agents, we have tried to isolate a cDNA clone for rat liver catalase. A recombinant clone, pMJ501, was isolated, of which cDNA insert specifically hybridized to catalase mRNA in hybridization-selected translation. On RNA blot hybridization, it hybridized to 2.4-kilobases RNA which was increased about 1.5-fold by the administration of di-(2-ethylhexyl)phthalate to the rats. The nucleotide sequence of the cDNA contains a reading frame for 109 amino acid residues which match the reported amino acid sequence of bovine liver catalase at the carboxyl end with 82% homology. It is concluded that pMJ501 contains a cDNA sequence for rat liver catalase.

Purification and characterization of a rat liver ferroactivator with catalase activity.

A rat liver protein with both phosphoenolpyruvate carboxykinase ferroactivator activity and catalase activity has been purified to near-homogeneity. The protein has a native molecular weight of 240,000 and is composed of four identical subunits containing ferriprotoporphyrin IX prosthetic groups. The visible spectrum has absorbance maxima at 403, 500, 530, and 620 nm; it is not reduced by dithionite. The spectrum, physical properties, and specific activity are almost identical with those of catalases from other sources, and the protein has been tentatively identified as rat liver catalase. The protein exhibited partial reactivity in double immunodiffusion plates to antiserum prepared against rat liver ferroactivator isolated by a previous method (Bentle, L. A., and Lardy, H. A. (1977) J. Biol. Chem. 252, 1431-1440) raising the possibility that the original ferroactivator and rat liver catalase are structurally related. Inactivation of catalase by 3-amino-1,2,4-triazole was accompanied by loss of ferroactivator activity as well. The apparent specific activity of ferroactivator, as well. The apparent specific activity of ferroactivator, whether heme-containing or not, can be increased between 2- and 100-fold by the inclusion of bovine serum albumin, HCO3-, or a combination of the two in the incubation.

Immunocytochemical localization of catalase in rat liver.

The intracellular localization of catalase has been studied using monospecific Fab fragments against rat liver catalase (RLC) and preembedding immunoelectron microscopy. Purified RLC, exhibiting a single band on sodium dodecyl sulfate gel electrophoresis, was used for the immunization of rabbits. The anti-RLC IgG was purified by affinity chromatography. Fab fragments were obtained by papain digestion and were labeled with horseradish peroxidase (HRP) using a modified two-step procedure and glutaraldehyde as coupling agents. Livers were perfused with 4% depolymerized paraformaldehyde and chopper sections were incubated with HRP-labeled Fab fragments against RLC. Because of the limited penetration of labeled Fab fragments into chopper sections a simple method for preparation of a cell suspension from aldehyde-fixed livers was devised. Adequate staining of more than 90% of cell was obtained by incubation of cell suspensions for 12--18 hr with the labeled antibody. By light microscopy specific staining was present in fine granules in the cytoplasm of hepatocytes. By electron microscopy the electron-dense reaction product was localized in the matrix of peroxisomes with no reaction in the endoplasmic reticulum and the Golgi complex. In some hepatocytes, positively reacted peroxisomes were seen side by side with unstained particles. Although focal diffusion was noted around a few peroxisomes, no evidence of cytoplasmic catalase independent of peroxisomes was found. These observations indicate that in rat liver peroxisomes are the only organelle containing substantial amounts of catalase antigen and rule out any involvement of the endoplasmic reticulum and the Golgi complex in the sequestration of this protein.

Selective cytochemical localization of peroxidase, cytochrome oxidase and catalase in rat liver with 3,3?-diaminobenzidine

In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specificity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15-30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylte buffer pH 6.5 and 0.02% H2O2. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) buffer at pH 10.5 and 0.15% H2O2. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.

Studies on rat liver catalase. XI. Site of synthesis and segregation by stripped ER membranes.

We reinvestigated the site of synthesis of rat liver catalase, and it has been reconfirmed that catalase is synthesized not only by free polysomes but also by membrane-bound polysomes. Considerable amounts of nascent catalase on rough microsomes were released from the membrane into the medium upon incubation with puromycin, not transported directly into the intracisternal cavity of microsomes. On the other hand, catalase newly synthesized in vitro was shown to be segregated by stripped rat liver microsomal membranes in a state resistant to proteolysis. Since this segregation occurred without coupled protein synthesis, catalase appears to be transported by a mechanism different from co-translational transfer. A hypothesis is presented regarding the mechanism of intracellular transport of liver catalase.

Studies on Rat Liver Catalase

Rat liver catalase mRNA was translated in a rabbit reticulocyte lysates and wheat germ cell-free system in the presence or absence of hemin and/or a translational inhibitor prepared from reticulocytes, liver cells, and wheat germs. Failure to add hemin to the lysates, or the addition of a hemin-regulated translational inhibitor (HRI) to the hemin-supplemented lysates caused a repressed translation. A preparation of inhibitor from rat liver showed activity similar to that of HRI for this translating system. The translation repression by rat liver inhibitor was reversed by eIF-2 (initiation factor) or GTP, but ATP enhanced the repression. The translation of catalase mRNA in the wheat germ system was not affected by the addition of hemin. An inhibitor prepared from wheat germ extracts, as well as the rat liver inhibitor, markedly decreased the rate of translation. eIF-2, GTP, and ATP behaved in the manner described above. Catalase synthesis in a cell-free system derived from rat liver (using endogenous mRNA) was not influenced by either hemin or the inhibitor. The possibilities are discussed that the synthesis of catalase in liver cells is controlled by a translational inhibitor at the level of chain initiation, and that the formation of the inhibitor from its inactive proinhibitor is regulated by the amount of heme.

Enhancement of rat liver catalase activity dietary cholesterol

The effects of a fat-supplemented diet and clofibrate (ethylchlorophenoxyisobutirate) upon serum lipids and liver catalase activity were studied in male rats. A butter-supplemented diet produced a striking increase of serum triglycerides but did not affect the liver catalase activity. Cholesterol (1%, w/w), added to the butter supplemented diet markedly increased liver catalase activity. This diet produced a hypercholesterolemic state higher than that induced by a butter-supplemented diet only, although the hypertriglyceridemic effect was less pronounced. Clofibrate given a butter-supplemented diet produced a marked increase of liver catalase activity (about four-fold). When clofibrate is administered with the cholesterol-supplemented diet, the increment observed in the liver catalase activity was the same as that induced with the cholesterol supplemented diet alone. Clofibrate, in either lipid-rich diet, failed to induce a hypocholesterolemic response, although a clear hypotrigliceridemic effect was evident. This effect appears to be potentiated with clofibrate and the cholesterol supplemented diet. Thus the increment in liver catalase activity induced by dietary cholesterol and clofibrate seems to be related to a hypotriglyceridemic effect which gives support to a role of liver peroxisomes in lipid metabolism. The role that liver catalase would play, in this regard, remains unclear from these results.

Tritium effect in peroxidation of ehtanol by liver catalase.

1. Simultaneous determination of the rate of appearance of 3H in water from [(1R)-1-3H1] ethanol and the rate of acetaldehyde formation in the presence of rat or ox liver catalase under conditions of steady-state generation of H2O2 allowed calculation of the 3H isotope effect. The mean value of 2.52 obtained for rat liver catalase at 37 degrees C and pH 6.3-7.7 was independent of both ethanol concentration and the rate of H2O2 generation over a wide range. At 25 degrees C a slightly lower mean value of 2.40 was obtained with the ox liver catalase. 2. Neither the product, acetaldehyde, nor 4-methylpyrazole influenced the two rates measured in the assay. 3. Relating the value obtained for the 3H isotope effect to a known value for the 2H isotope effect strongly supports the view that both values are close to the true isotope effect with the respective substituted compounds on the rate constant in the catalytic step involving scission of the C-H bond. 4. The constancy of the isotope effect under various conditions makes it possible to use it for interpretations in vivo. 5. It was established that beta-D-galactose dehydrogenase exhibits B-specificity towards the nicotinamide ring in NAD.

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Initiation with methionine of the synthesis of rat liver catalase [EC 1.11.1.6] has been investigated. Analysis of the N-terminal residue of nascent catalase peptides labeled in vivo with injected radioactive amino acids, including [3H]methionine, indicated a remarkably high content of methionine. By fractionating [3H]methionine-labeled nascent catalase according to chain length, it was found that peptides of shorter chain length contained more N-terminal methionine relative to total methionine incorporated. In addition, only a small amount of [3H]methionine was detected as the N-terminal amino acid when newly completed catalase was examined by Edman degradation. These results indicate that the synthesis of liver catalase is initiated with methionine, and suggest the presence of a mechanism for its subsequent removal from the N-terminal position. Catalase was also synthesized in a cell-free system directed by the catalase mRNA, using [3H]Met-tRNAf or [3H]Met-tRNAm. The results obtained in such in vitro experiments were in good agreement with those from in vivo studies, and further showed that the N-terminal methionine was provided by a specific initiator tRNA, i.e. tRNA Met f.