Cytochemical detection of catalase with 3,3′-diaminobenzidine

Summary The influence of various parameters of fixation and incubation upon the oxidation of DAB by catalase have been analyzed. Crystalline beef liver catalase was fixed with different concentrations of glutaraldehyde and peroxidatic activity was determined spectrophotometrically using DAB as hydrogen donor. Although aldehyde fixation appeared to be important in elicitation of the peroxidatic activity of catalase, the final pigment production after 60 min incubation was optimal with the lowest concentration of glutaraldehyde (1%), after the shortest fixation period (30 min), and at the lowest temperature (5° C) tested. Similarly cytochemical studies with rat kidney sections incubated for 10 min confirmed that the staining of peroxisomes in proximal tubules was strongest after the mildest fixation conditions. The pH and the temperature of incubation were closely interrelated, so that at room temperature (25° C) the maximal pigment production was obtained at pH 10.5 but incubation at 45° C gave the strongest staining at pH 8.5. The production of pigment increased with higher DAB concentrations which required larger amounts of H₂O₂ in the incubation medium. Cytochemical studies on renal peroxisomes were in agreement with these biochemical findings. The observations indicate that there are several options for the localization of catalase depending on the fixation and incubation conditions. Hence, these conditions should be selected according to the tissue and the purpose of the study. Examples for such selective applications are presented.     

γ-Butyrobetaine hydroxylase: A unique protective effect of catalase

Catalase stimulates the activity of homogeneous γ-butyrobetaine hydroxylase by approximately 300-fold. The stimulation of the hydroxylation reaction elicited by catalase is saturable, and although a number of proteins may be substituted for catalase, none is as effective. γ-Butyrobetaine hydroxylase is also irreversibly inactivated in the presence of one of its substrates, oxygen, and its cofactor, ascorbate. This inactivation of the hydroxylase activity may be prevented by (i) the presence of high concentrations (2 mg/ml) of various proteins, (ii) the presence of catalytic concentrations (20 μg/ml) of catalase, or (iii) the presence of 10 mm histidine or dithiothreitol. Oxidized species of ascorbate do not appear to be responsible for the inactivation process. Time-dependent inactivation is also observed when γ-butyrobetaine hydroxylase is preincubated with hydrogen peroxide generated by the glucose oxidase-catalyzed oxidation of glucose. At low concentrations, superoxide dismutase was not as effective as an equivalent protein concentration of catalase in protecting against inactivation, and hydroxyl radical scavengers were completely ineffective. In measurements of γ-butyrobetaine hydroxylase activity, the presence of catalase both stimulates the catalytic activity of the hydroxylase and protects the enzyme from inactivation by a product of the interaction of components in the assay mixture, presumably hydrogen peroxide.     

Facile synthesis of peptide–porphyrin conjugates: Towards artificial catalase

A facile synthetic method for peptide–porphyrin conjugates containing four peptide units on one porphyrin was developed using chemoselective reactions. The key building blocks, 5,10,15,20-tetrakis(3-azidophenyl)porphyrin 1 and 5,10,15,20-tetrakis(5-azido-3-pyridyl)porphyrin 2, were efficiently synthesized and used as substrates for two well-known chemoselective reactions, traceless Staudinger ligation and copper-catalyzed azide alkyne cycloaddition (so-called click chemistry). Both reactions gave the desired compounds, and click chemistry was superior for our purpose. To confirm the value of the established methodology, nine peptide–porphyrin conjugates were synthesized, and their catalase- and peroxidase-like activity in water was evaluated. Our synthetic strategy is expected to be valuable for the preparation of artificial heme protein models.     

Probing the two-domain structure of homodimeric prokaryotic and eukaryotic catalase–peroxidases

Catalase–peroxidases (KatGs) are ancestral bifunctional heme peroxidases found in archaeons, bacteria and lower eukaryotes. In contrast to homologous cytochrome c peroxidase (CcP) and ascorbate peroxidase (APx) homodimeric KatGs have a two-domain monomeric structure with a catalytic N-terminal heme domain and a C-terminal domain of high sequence and structural similarity but without obvious function. Nevertheless, without its C-terminal counterpart the N-terminal domain exhibits neither catalase nor peroxidase activity. Except some hybrid-type proteins all other members of the peroxidase–catalase superfamily lack this C-terminal domain. In order to probe the role of the two-domain monomeric structure for conformational and thermal stability urea and temperature-dependent unfolding experiments were performed by using UV–Vis-, electronic circular dichroism- and fluorescence spectroscopy, as well as differential scanning calorimetry. Recombinant prokaryotic (cyanobacterial KatG from Synechocystis sp. PCC6803) and eukaryotic (fungal KatG from Magnaporthe grisea) were investigated. The obtained data demonstrate that the conformational and thermal stability of bifunctional KatGs is significantly lower compared to homologous monofunctional peroxidases. The N- and C-terminal domains do not unfold independently. Differences between the cyanobacterial and the fungal enzyme are relatively small. Data will be discussed with respect to known structure and function of KatG, CcP and APx.     

Dopamine-β-hydroxylase: Stimulation by nitrogen-containing heterocyclics and the role of catalase

The catalase inhibitor 3-amino-1,2,4-triazole causes an increase in dopamine--hydroxylase (DBH) activity, as do other nitrogen-containing heterocyclics. Denatured catalase also causes an increase in activity, but in both cases, optimum activity is attained only in the presence of some native catalase. It is proposed that the latter affects the DBH reaction in two different ways: It decomposes toxic peroxides, and it stabilizes the enzyme in some manner as yet unknown, as do the heterocyclics. The nitrogen-containing compounds, and denatured catalase, protect DBH from inhibition by copper. Ideas concerning the relationships of copper, catalase, and DBH must be altered to accommodate these new data.This study was supported in part by United States Public Health Service Grant NS-04454. A preliminary report of these experiments was presented at a Symposium on New First and Second Messengers in Nervous Tissues in Brescia, Italy, August 28–30, 1975.     

Peroxisome proliferator-activated receptor γ regulates angiotensin II-induced catalase downregulation in adventitial fibroblasts of rats

Peroxisome proliferator-activated receptor (PPAR) γ ligands oppose the effect induced by angiotensin II (Ang II) to reduce oxidative stress and improve antioxidant status. In this study, Ang II inhibited catalase (CAT) and peroxisome proliferator-activated receptor γ (PPAR γ) protein and mRNA expressions. Transfection with PPAR γ small-interfering RNA (siRNA) led to a reduction in CAT expression. PPAR γ ligands enhanced CAT expression and inhibited extracellular signal-regulated kinase 1/2 activation. We further reveal that Ang II type 1 receptor is not involved in the inhibitory effects of PPAR γ ligands on Ang II stimulatory events.     

Activation of catalase activity by a peroxisome-localized small heat shock protein Hsp17.6CⅡ

Plant catalases are important antioxidant enzymes and are indispensable for plant to cope with adverse environmental stresses. However, little is known how catalase activity is regulated especially at an organelle level. In this study, we identified that small heat shock protein Hsp17.6CⅡ(AT5G12020) interacts with and activates catalases in the peroxisome of Arabidopsis thaliana. Although Hsp17.6CⅡ is classified into the cytosol-located small heat shock protein subfamily, we found that Hsp17.6CⅡ is located in the peroxisome. Moreover, Hsp17.6CⅡ contains a novel non-canonical peroxisome targeting signal 1(PTS1), QKL, 16 amino acids upstream from the C-terminus. The QKL signal peptide can partially locate GFP to peroxisome, and mutations in the tripeptide lead to the abolishment of this activity. In vitro catalase activity assay and holdase activity assay showed that Hsp17.6CⅡ increases CAT2 activity and prevents it from thermal aggregation. These results indicate that Hsp17.6CⅡ is a peroxisome-localized catalase chaperone. Overexpression of Hsp17.6CⅡ conferred enhanced catalase activity and tolerance to abiotic stresses in Arabidopsis. Interestingly, overexpression of Hsp17.6CⅡ in catalase-deficient mutants,nca1-3 and cat2 cat3, failed to rescue their stress-sensitive phenotypes and catalase activity, suggesting that Hsp17.6CⅡ-mediated stress response is dependent on NCA1 and catalase activity. Overall, we identified a novel peroxisome-located catalase chaperone that is involved in plant abiotic stress resistance by activating catalase activity.     

Is catalase activity one of the factors associated with maize resistance to Aspergillus flavus?

Plant responses to biotic and abiotic stresses are usually accompanied by the release of reactive oxygen species including hydrogen peroxide. Hydrogen peroxide plays a direct role in defense and is involved in many signal transduction pathways that lead to the proliferation of other defenses. Because catalase helps to maintain reactive oxygen homeostasis during biotic and abiotic stress, its activity was measured in various cob tissues during maize ear development. Catalase activity was determined in immature and mature embryos, pericarp, and rachis tissues of maize lines that are resistant and susceptible to Aspergillus flavus infection. The effect of fungal inoculation on catalase activity was also measured. Over two years of field experimentation, a correlation was observed between resistance and the level of catalase-specific activity in immature embryos, which was significantly higher in resistant lines (P < 0.0001). Furthermore, catalase activity in the resistant lines was significantly higher in immature embryos from inoculated ears (P = 0.0199). No correlation was observed between resistance and catalase activity in other ear tissues. Levels of hydrogen peroxide, the catalase substrate, and salicylic acid in the embryo were also determined. The resistant lines showed lower levels of H2O2 (P < 0.0001) and higher levels of salicylic acid (P < 0.0001) as compared with the susceptible lines. Catalase 3 was sequenced from the aflatoxin-resistant (Mp313E) and susceptible (SC212m) inbreds. The predicted amino acid sequence indicated that there was a 20-aa deletion in the resistant inbred that might affect enzymatic activity. Unlike many plant-pathogen interactions, it appears that lowering H2O2 levels helps to prevent A. flavus infection and subsequent aflatoxin accumulation.     

The mechanism of photoinhibition in vivo: Re-evaluation of the roles of catalase, α-tocopherol, non-photochemical quenching, and electron transport

Photoinhibition of photosystem II (PSII) occurs when the rate of light-induced inactivation (photodamage) of PSII exceeds the rate of repair of the photodamaged PSII. For the quantitative analysis of the mechanism of photoinhibition of PSII, it is essential to monitor the rate of photodamage and the rate of repair separately and, also, to examine the respective effects of various perturbations on the two processes. This strategy has allowed the re-evaluation of the results of previous studies of photoinhibition and has provided insight into the roles of factors and mechanisms that protect PSII from photoinhibition, such as catalases and peroxidases, which are efficient scavengers of H(2)O(2); α-tocopherol, which is an efficient scavenger of singlet oxygen; non-photochemical quenching, which dissipates excess light energy that has been absorbed by PSII; and the cyclic and non-cyclic transport of electrons. Early studies of photoinhibition suggested that all of these factors and mechanisms protect PSII against photodamage. However, re-evaluation by the strategy mentioned above has indicated that, rather than protecting PSII from photodamage, they stimulate protein synthesis, with resultant repair of PSII and mitigation of photoinhibition. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Promoter engineering of cascade biocatalysis for α-ketoglutaric acid production by coexpressing l-glutamate oxidase and catalase

Applied Microbiology and Biotechnology - Enzymatic transformation is now an attractive alternative for α-ketoglutaric acid (α-KG) production, but the oxidative deamination from l-glutamic...     

Regulation of the oxidative stress protective enzymes,catalase and superoxide dismutase in Xanthomonas — a review

Xanthomonas showed atypical regulation of catalase (Kat) and superoxide dismutase with respect to growth phase and response to various inducers. The highest levels of both enzymes were detected during early log phase of growth and declined as growth continued. This was in contrast to resistance levels to superoxides, H₂O₂ and organic peroxides, which reached maximum levels during stationary phase. Xanthomonas catalase was induced over six fold by superoxide generators and methyl methane sulfonate but weakly by H₂O₂. The regulation pattern of these enzymes could be important during plant/microbe interactions. To facilitate elucidation of Xanthomonas kat gene regulation, highly conserved regions of monofuctional Kat amino acid sequences were used to synthesize oligodeoxyribonucleotide primers for use in PCR reactions with Xanthomonas genomic DNA as templates. The Xanthomonas-specific PCR kat probe was used to isolate a functional kat from Xanthomonas campestris pv. phaseoli.     

Cardioprotection from oxidative stress in the newborn heart by activation of PPARγ is mediated by catalase

Regulation of catalase (CAT) by peroxisome proliferator-activated receptor-γ (PPARγ) was investigated to determine if PPARγ activation provides cardioprotection from oxidative stress caused by hydrogen peroxide (H(2)O(2)) in an age-dependent manner. Left ventricular developed pressure (LVDP) was measured in Langendorff perfused newborn or adult rabbit hearts, exposed to 200μM H(2)O(2), with perfusion of rosiglitazone (RGZ) or pioglitazone (PGZ), PPARγ agonists. We found: (1) H(2)O(2) significantly decreased sarcomere shortening in newborn ventricular cells but not in adult cells. Lactate dehydrogenase (LDH) release occurred earlier in newborn than in adult heart, which may be due, in part, to the lower expression of CAT in newborn heart. (2) RGZ increased CAT mRNA and protein as well as activity in newborn but not in adult heart. GW9662 (PPARγ blocker) eliminated the increased CAT mRNA by RGZ. (3) In newborn heart, RGZ and PGZ treatment inhibited release of LDH in response to H(2)O(2) compared to H(2)O(2) alone. GW9662 decreased this inhibition. (4) LVDP was significantly higher in both RGZ+H(2)O(2) and PGZ+H(2)O(2) groups than in the H(2)O(2) group. Block of PPARγ abolished this effect. In contrast, there was no effect of RGZ in adult. (5) The cardioprotective effects of RGZ were abolished by inhibition of CAT. In conclusion, PPARγ activation is cardioprotective to H(2)O(2)-induced stress in the newborn heart by upregulation of catalase. These data suggest that PPARγ activation may be an effective therapy for the young cardiac patient.     

How does the push/pull effect of the axial ligand influence the catalytic properties of Compound I of catalase and cytochrome P450?

Density functional theory (DFT) calculations on the chemoselective epoxidation versus hydroxylation reactions of propene by oxoiron porphyrin models mimicking the active sites of catalase, cytochrome P450 (P450) and horseradish peroxidase Compound I (CpdI) are presented. The catalase reactions are concerted and proceed via two-state reactivity patterns on competing doublet and quartet spin state surfaces, but the lowest barrier is the one leading to epoxide products on the doublet spin surface. The results are compared with earlier DFT studies of models of cytochrome P450, horseradish peroxide (HRP), taurine/alpha-ketoglutarate dioxygenase and some synthetic oxoiron catalysts. The catalase barriers are midway in between those obtained for HRP and P450 models, so that tyrosinate ligated heme systems should be able to catalyze C-H hydroxylation and C=C epoxidation reactions. We show that for heme systems the barrier height of epoxidation linearly correlates with the electron affinity of Compound I as expected from the electron transfer mechanism of the rate determining step. Our studies show that the axial ligand does not influence the chemoselectivity of a reaction but that it does regulate the barrier heights and rate constants. Finally, we estimated the effect of the axial ligand on the oxoiron group and derived that it contributes from a field effect due to the charge of the ligand and a quantum mechanical effect as a result of orbital mixing. In catalase, the major component is the field effect, while the quantum mechanical effect is negligible. This is in contrast to P450 CpdI, where both effects are of similar order of magnitude.     

Oxidation of phenolic compounds by the bifunctional catalase–phenol oxidase (CATPO) from Scytalidium thermophilum

The thermophilic fungus Scytalidium thermophilum produces a novel bifunctional catalase with an additional phenol oxidase activity (CATPO); however, its phenol oxidation spectrum is not known. Here, 14 phenolic compounds were selected as substrates, among which (+)-catechin, catechol, caffeic acid, and chlorogenic acid yielded distinct oxidation products examined by reversed-phase HPLC chromatography method. Characterization of the products by LC-ESI/MS and UV–vis spectroscopy suggests the formation of dimers of dehydrocatechin type B (hydrophilic) and type A (hydrophobic), as well as oligomers, namely, a trimer and tetramer from (+)-catechin, the formation of a dimer and oligomer of catechol, a dimer from caffeic acid with a caffeicin-like structure, as well as trimeric and tetrameric derivatives, and a single major product from chlorogenic acid suggested to be a dimer. Based on the results, CATPO oxidizes phenolic compounds ranging from simple phenols to polyphenols but all having an ortho-diphenolic structure in common. The enzyme also appears to have stereoselectivity due to the oxidation of (+)-catechin, but not that of epicatechin. It is suggested that CATPO may contribute to the antioxidant mechanism of the fungus and may be of value for future food and biotechnology applications where such a bifunctional activity would be desirable.     

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

Summary 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 specifity 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 cacodylate 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) butffer 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.     

Dimanganese catalase—spectroscopic parameters from broken-symmetry density functional theory of the superoxidized MnIII/MnIV state

Broken-symmetry density functional theory was used to study the catalytic center of manganese catalase in the superoxidized Mn^III/Mn^IV state. Heisenberg exchange coupling constants, ⁵⁵Mn and ¹⁴N hyperfine coupling constants (hfcs) and nuclear quadrupole splittings, as well as the electronic g tensors were evaluated for different model systems of the active site after complete geometry optimizations in the high-spin and broken-symmetry states. A comparison of the experimental data with the spectroscopic parameters computed for the models with unprotonated and protonated -oxo bridges shows best agreement between theory and experiment for a Mn₂(-O)₂(-OAc) core. The calculated Mn–Mn distances and ⁵⁵Mn hfcs clearly support a dimanganese cluster with unprotonated -oxo bridges in the superoxidized state. Furthermore, it is shown that an interchange of the Mn^III and Mn^IV oxidation states in this trapped valence system leads to specific changes in the molecular and electronic structure of the manganese clusters.     

Integral role of the I′-helix in the function of the “inactive” C-terminal domain of catalase–peroxidase (KatG)

Catalase–peroxidases (KatGs) have two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. Though the C-terminal domain lacks the ability to bind heme or directly catalyze any reaction, it has been proposed to serve as a platform to direct the folding of the N-terminal domain. Toward such a purpose, its I′-helix is highly conserved and appears at the interface between the two domains. Single and multiple substitution variants targeting highly conserved residues of the I′-helix were generated for intact KatG as well as the stand-alone C-terminal domain (KatG^C). Single variants of intact KatG produced only subtle variations in spectroscopic and catalytic properties of the enzyme. However, the double and quadruple variants showed substantial increases in hexa-coordinate low-spin heme and diminished enzyme activity, similar to that observed for the N-terminal domain on its own (KatG^N). The analogous variants of KatG^C showed a much more profound loss of function as evaluated by their ability to return KatG^N to its active conformation. All of the single variants showed a substantial decrease in the rate and extent of KatG^N reactivation, but with two substitutions, KatG^C completely lost its capacity for the reactivation of KatG^N. These results suggest that the I′-helix is central to direct structural adjustments in the adjacent N-terminal domain and supports the hypothesis that the C-terminal domain serves as a platform to direct N-terminal domain conformation and bifunctionality.     

Expression of a cloned sweet potato catalase SPCAT1 alleviates ethephon-mediated leaf senescence and H₂O₂ elevation

In this report a full-length cDNA, SPCAT1, was isolated from ethephon-treated mature L3 leaves of sweet potato. SPCAT1 contained 1479 nucleotides (492 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 71.2-80.9%) with several plant catalases, including Arabidopsis, eggplant, grey mangrove, pea, potato, tobacco and tomato. Gene structural analysis showed that SPCAT1 encoded a catalase and contained a putative conserved internal peroxisomal targeting signal PTS1 motif and calmodulin binding domain around its C-terminus. RT-PCR showed that SPCAT1 gene expression was enhanced significantly in mature L3 and early senescent L4 leaves and was much reduced in immature L1, L2 and completely yellowing senescent L5 leaves. In dark- and ethephon-treated L3 leaves, SPCAT1 expression was significantly enhanced temporarily from 0 to 24 h, then decreased gradually until 72 h after treatment. SPCAT1 gene expression levels also exhibited approximately inverse correlation with the qualitative and quantitative H₂O₂ amounts. Effector treatment showed that ethephon-enhanced SPCAT1 expression was repressed by antioxidant reduced glutathione, NADPH oxidase inhibitor diphenylene iodonium (DPI), calcium ion chelator EGTA and de novo protein synthesis inhibitor cycloheximide. These data suggest that elevated reactive oxygen species H₂O₂, NADPH oxidase, external calcium influx and de novo synthesized proteins are required and associated with ethephon-mediated enhancement of sweet potato catalase SPCAT1 expression. Exogenous application of expressed catalase SPCAT1 fusion protein delayed or alleviated ethephon-mediated leaf senescence and H₂O₂ elevation. Based on these data we conclude that sweet potato SPCAT1 is an ethephon-inducible peroxisomal catalase, and its expression is regulated by reduced glutathione, DPI, EGTA and cycloheximide. Sweet potato catalase SPCAT1 may play a physiological role or function in cope with H₂O₂ homeostasis in leaves caused by developmental cues and environmental stimuli.     

The relationship between δ-aminolaevulinate synthetase induction and the concentrations of cytochrome P-450 and catalase in rat liver

The porphyrinogenic drug 2-allyl-2-isopropylacetamide causes the degradation of microsomal cytochrome P-450 and inhibits the synthesis of catalase in rat liver. The inhibition of catalase synthesis follows the induction of delta-aminolaevulinate synthetase and the consequent overproduction of haem. The allylisopropylacetamide-mediated breakdown of cytochrome P-450 is a rapid event and has a reciprocal relationship to the pattern of delta-aminolaevulinate synthetase induction. Breakdown of cytochrome P-450 appears to be one of the conditions leading to the ;derepression' of delta-aminolaevulinate synthetase.