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PubMed1964

CYTOCHEMICAL OBSERVATIONS ON THE RELATIONSHIP BETWEEN LYSOSOMES AND PHAGOSOMES IN KIDNEY AND LIVER BY COMBINED STAINING FOR ACID PHOSPHATASE AND INTRAVENOUSLY INJECTED HORSERADISH PEROXIDASE.

After incubation of formalin-fixed, frozen sections of kidney and liver from peroxidase-treated rats in an azo dye medium for acid phosphatase, and after subsequent incubation of the same sections with benzidine, phagosomes were stained blue and lysosomes were stained red in the same cells. It was observed that newly formed phagosomes were separate from preexisting lysosomes in the tubule cells of the kidney and in the Kupffer cells of the liver at early periods after treatment with peroxidase. At later periods, the color reactions for acid phosphatase and peroxidase occurred in the same granules. The reaction of peroxidase decreased gradually and disappeared from the phago-lysosomes after 2 to 3 days, whereas the reaction for acid phosphatase persisted. In the liver, most of the injected protein was concentrated in large phagosomes located at the periphery of the cells lining the sinusoids. The peribiliary lysosomes showed a relatively weak reaction for peroxidase in the proximity of the portal veins. After pathological changes of permeability, phagosomes and lysosomes lost their normal location and fused, in the interior of many liver cells, to form large vacuoles or spheres. The effects of a reduced load of peroxidase and the effects of the pretreatment with another protein (egg white) on the phago-lysosomes of the kidney were tested. The relationship of the fusion of phagosomes with lysosomes to the size of normal and pathological phago-lysosomes was discussed.

Acid Phosphatase↗
PubMed2001

Iron deficiency differently affects peroxidase isoforms in sunflower.

The response of both specific (ascorbate peroxidase, APX) and unspecific (POD) peroxidases and H(2)O(2) content of sunflower plants (Helianthus annuus L. cv. Hor) grown hydroponically with (C) or without (-Fe) iron in the nutrient solution were analysed to verify whether iron deficiency led to cell oxidative status. In -Fe leaves a significant increase of H(2)O(2) content was detected, a result confirmed by electron microscopy analysis. As regards extracellular peroxidases, while APX activity significantly decreased, no change was observed in either soluble guaiacol or syringaldazine-dependent POD activity following iron starvation. Moreover, guaiacol-dependent POD activity was found to decrease in both ionically and covalently-cell-wall bound fractions, while syringaldazine-POD activity decreased only in the covalently-bound fraction. At the intracellular level both guaiacol-POD and APX activities underwent a significant decrease. The overall reduction of peroxidase activity was confirmed by the electrophoretic separation of POD isoforms and, at the extracellular level, by cytochemical localization of peroxidases by diaminobenzidine staining. The electrophoretic separation, besides quantitative differences, also revealed quantitative changes, particularly evident for ionically and covalently-bound fractions. Therefore, in sunflower plants, iron deficiency seems to affect the different peroxidase isoenzymes to different extents and to induce a secondary oxidative stress, as indicated by the increased levels of H(2)O(2). However, owing to the almost completely lack of catalytic iron capable of triggering the Fenton reaction, iron-deficient sunflower plants are probably still sufficiently protected against oxidative stress.

Ascorbate Peroxidases↗
PubMed1989

Molecular cloning and characterization of a chromosomal gene for human eosinophil peroxidase.

Using human myeloperoxidase cDNA as a probe, a chromosomal gene related to myeloperoxidase was isolated from a human gene library. Comparison of the amino acid sequence deduced from the nucleotide sequence of the cloned gene with that of human eosinophil peroxidase purified from buffy coats has indicated that the isolated gene is the chromosomal gene for human eosinophil peroxidase. Like human myeloperoxidase gene, human eosinophil peroxidase gene consists of 12 exons and 11 introns spanning about 12 kilobases. The gene can code for a protein of 715 amino acids with a calculated Mr of 81,036. The heavy chain and the light chain of eosinophil peroxidase were located on the COOH and NH2 terminus of the protein, respectively. The coding sequences of eosinophil peroxidase and myeloperoxidase show homologies of 72.4% at the nucleotide and 69.8% at the amino acid level, while little homology was found in the 5'-flanking region. Northern hybridization and S1 mapping analysis of RNA from human leukemic cells have indicated that the eosinophil peroxidase gene is expressed in the eosinophilic subline of human HL-60 cells but not in the neutrophilic subline or in parental HL-60 cells.

Amino Acid Sequence↗
PubMed2001

Effect of reaction conditions on phenol removal by polymerization and precipitation using Coprinus cinereus peroxidase.

The quantitative relationships between removal efficiency of phenol and reaction conditions were investigated using Coprinus cinereus peroxidase. The most effective ratio of hydrogen peroxide to phenol was nearly 1/1 (mol/mol) at an adequate enzyme dose. 12.2 U of the enzyme was needed to remove 1 mg of phenol when our peroxidase preparation was used. At an insufficient peroxidase dose, the optimum pH value was 9.0, and lowering the reaction temperature led to the improvement of removal efficiency. At an excess peroxidase dose, almost 100% removal of phenol was obtained over a wide range of pH (5-9) and temperature (0-60 degrees C). Despite the presence of culture medium components, it was shown that Coprinus cinereus peroxidase had the same phenol polymerization performance as horseradish peroxidase or Arthromyces ramosus peroxidase.

Journal Article↗
PubMed2000

Glycosylation of the cationic peanut peroxidase gene expressed in transgenic tobacco.

The major cationic peanut (Arachis hypogaea) peroxidase, secreted into the extracellular space, is a glycoprotein with three N-linked glycans (polysaccharides) which are connected to the peptide backbone at Asn-60, Asn-144 and Asn-185. In this report, a C-terminal histidine-tagged cationic peanut peroxidase gene was expressed in transgenic tobacco (Nicotiana tabacum). Tissue of the transgenic tobacco was cultured in suspension culture and the his-tagged peroxidase was purified in large quantities from 14-day-old suspension culture. The number of glycans, glycosylation sites and the chemical nature of glycan moieties attached to cationic peanut peroxidase expressed in transgenic tobacco were examined. Cationic peanut peroxidase isolated from the above transgenic tobacco had the identical number of complex glycans, attached at the same glycosylation sites as on cationic peanut peroxidase isolated from peanut suspension culture. Monosaccharide components of these glycans are N-acetylglucosamine (GlcNAc), mannose (Man), fucose (Fuc), xylose (Xyl) and galactose (Gal), the same sugars as found in native cationic peanut peroxidase.

Journal Article↗
PubMed2002

Conversion of a typical catalase from Bacillus sp. TE124 to a catalase-peroxidase by directed evolution.

We have converted a typical catalase from Bacillus sp. TE124 to a catalase-peroxidase using DNA shuffling and error-prone PCR. A triple mutant, R47H/R356C/D374N, that showed significantly reduced catalase activity and increased peroxidase activity was identified by screening mutant libraries. When single mutant--R47H, R356C and D374N--were generated by site-directed mutagenesis, conserved Arg-47, located on the distal side of the prosthetic heme group in the superfamily of typical catalases, was found to be responsible for the conversion of catalase to catalase-peroxidase. To further clarify the role of Arg-47, arginine was replaced with different amino acids--alanine, lysine, aspartic acid, glutamic acid, glutamine, phenylalanine, tryptophan and tyrosine--and the mutant enzymes were assayed. All of the arginine mutants had increased peroxidase activity coupled with reduced catalase activity. Among these mutants, R47W exhibited the highest peroxidase activity, while R47E and R47Q not only had increased peroxidase activity but also retained relatively high catalase activity. These results suggest that tryptophan plays a key role in the catalytic mechanism of the peroxidase reaction and that glutamic acid and glutamine facilitate both catalatic and peroxidatic reactions.

Journal Article↗
PubMed1991

Proton NMR investigation into the basis for the relatively high redox potential of lignin peroxidase.

Lignin peroxidase shares several structural features with the well-studied horseradish peroxidase and cytochrome c peroxidase but carries a higher redox potential. Here the heme domain of lignin peroxidase and the lignin peroxidase cyanide adduct was examined by 1HNMR spectroscopy, including nuclear Overhauser effect and two-dimensional measurements, and the findings were compared with those for horseradish peroxidase and cytochrome c peroxidase. Structural information was obtained on the orientation of the heme vinyl and propionate groups and the proximal and distal histidines. The shifts of the epsilon1 proton of the proximal histidine were found to be empirically related to the Fe3+/Fe2+ redox potentials.

Journal Article↗
PubMed1993

Crystal structure of lignin peroxidase.

The crystal structure of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium has been determined to 2.6 A resolution by usine multiple isomorphous replacement methods and simulated annealing refinement. Of the 343 residues, residues 3-335 have been accounted for in the electron density map, including four disulfide bonds. The overall three-dimensional structure is very similar to the only other peroxidase in this group for which a high-resolution crystal structure is available, cytochrome c peroxidase, despite the fact that the sequence identity is only approximately 20%, LiP has four disulfide bonds, while cytochrome c peroxidase has none, and LiP is larger (343 vs. 294 residues). The basic helical fold and connectivity defined by 11 helical segments with the heme sandwiched between the distal and proximal helices found in cytochrome c peroxidase is maintained in LiP. Both enzymes have a histidine as a proximal heme ligand, which is hydrogen bonded to a buried aspartic acid side chain. The distal or peroxide binding pocket also is similar, including the distal arginine and histidine. The most striking difference is that, whereas cytochrome c peroxidase has tryptophans contacting the distal and proximal heme surfaces, LiP has phenylalanines. This in part explains why, in the reaction with peroxides, cytochrome c peroxidase forms an amino acid-centered free radical, whereas LiP forms a porphyrin pi cation radical.

Journal Article↗
PubMed1981

A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies.

A highly sensitive immunoenzymatic technic is presented. The method involves three sequential steps: (1) primary antibody, (2) biotin-labeled secondary antibody, and (3) avidin-biotin-peroxidase complex. Avidin, an egg white protein, has four binding sites for the low-molecular-weight vitamin biotin. Many moieties of biotin can be coupled to the peroxidase molecule. Thus, since a relatively large amount of avidin is incubated with biotin-labeled peroxidase, avidin serves as a link between biotin-peroxidase molecules; in turn, biotin-peroxidase serves as a link between avidin molecules. Consequently, this large lattice-like complex with biotin-binding capability can be attracted to the sites of biotin-labeled antibody, producing a superior staining sensitivity. Several commercially available radioimmunoassay antibodies (e.g., antiglucagon, prolactin, gastrin, growth hormone, and thyroid-stimulating hormone antibodies) were tested for immunohistochemical staining. The unlabeled antibody peroxidase-antiperoxidase method fails to stain gastrin or thyroid-stimulating secretory cells when using these antibodies, and a relatively high antibody concentration is required to produce a positive reaction for glucagon, prolactin, and growth hormone. In contrast, the avidin-biotin-peroxidase complex method successfully demonstrates polypeptide hormones even when antibodies are diluted 20 to 40 times.

Antibodies↗
PubMed1995

Purification and Partial Characterization of Tomato Extensin Peroxidase.

Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as extensin, in the cell wall. The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases. We have ionically eluted extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculentum Mill. and Lycopersicon peruvianum L. [Mill.]) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography. Four ionic forms of peroxidase (PI,PII,EPIII, and EPIV) were resolved; only the latter two cross-linked tomato soluble extensin. The molecular weight (34,000-37,000), amino acid composition, and isoelectric point (9.0) of the extensin peroxidases were determined. Substrate specificities of the enzymes were investigated: soluble extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles extensin) were cross-linked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except extensin) could not be cross-linked. We have also isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge in cultured cells of tomato. A highly sensitive enzyme-linked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato extensin was used to demonstrate extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links extensin has been purified and may have a role in plant defense.

Journal Article↗
PubMed1997

Characterization of Antisense Transformed Plants Deficient in the Tobacco Anionic Peroxidase.

On the basis of the biological compounds that they metabolize, plant peroxidases have long been implicated in plant growth, cell wall biogenesis, lignification, and host defenses. Transgenic tobacco (Nicotiana tabacum L.) plants that underexpress anionic peroxidase were generated using antisense RNA. The antisense RNA was found to be specific for the anionic isoenzyme and highly effective, reducing endogenous transcript levels and total peroxidase activity by as much as 1600-fold. Antisense-transformed plants appeared normal at initial observation; however, growth studies showed that plants with reduced peroxidase activity grow taller and flower sooner than control plants. In contrast, previously transformed plants overproducing anionic peroxidase were shorter and flowered later than controls. Axillary buds were more developed in antisense-transformed plants and less developed in plants overproducing this enzyme. It was found that the lignin content in leaf, stem, and root was unchanged in antisense-transformed plants, which does not support a role for anionic peroxidase in the lignification of secondary xylem vessels. However, studies of wounded tissue show some reduction in wound-induced deposition of lignin-like polymers. The data support a possible role for tobacco anionic peroxidase in host defenses but not without a reduction in growth potential.

Journal Article↗
PubMed1973

Studies on Auxin Protectors: XI. Inhibition of Peroxidase-Catalyzed Oxidation of Glutathione by Auxin Protectors and o-Dihydroxyphenols.

Commercial horseradish peroxidase, when supplemented with dichlorophenol and either manganese or hydrogen peroxide, will rapidly oxidize glutathione. This peroxidase-catalyzed oxidation of glutathione is completely inhibited by the presence of auxin protectors. Three auxin protectors and three o-dihydroxyphenols were tested; all inhibited the oxidation. Glutathione oxidation by horseradish peroxidase in the presence of dichlorophenol and Mn is also completely inhibited by catalase, implying that the presence of Mn allows the horseradish peroxidase to reduce oxygen to H(2)O(2), then to use the H(2)O(2) as an electron acceptor in the oxidation of glutathione. Catalase, added 2 minutes after the glutathione oxidation had begun, completely inhibited further oxidation but did not restore any gluthathione oxidation intermediates. In contrast, the addition of auxin protectors, or o-dihydroxyphenols, not only inhibited further oxidation of gluthathione by horseradish peroxidase (+ dichlorophenol + Mn), but also caused a reappearance of glutathione as if these antioxidants reduced a glutathione oxidation intermediate. However, when gluthathione was oxidized by horseradish peroxidase in the presence of dichlorophenol and H(2)O(2) (rather than Mn), then the inhibition of further oxidation by auxin protectors or o-dihydroxyphenols was preceded by a brief period of greatly accelerated oxidation. The data provide further evidence that auxin protectors are cellular redox regulators. It is proposed that the monophenol-diphenol-peroxidase system is intimately associated with the metabolic switches that determine whether a cell divides or differentiates.

Journal Article↗
PubMed1979

Tomato peroxidase: purification, characterization, and catalytic properties.

A major peroxidase has been found in the tomato pericarp (Lycopersicon esculentum var. Tropic) of the ripe and green fruit. A purification scheme yielding this enzyme approximately 85% pure has been developed. The tomato enzyme resembles horseradish peroxidase (HRP) in a standard peroxidase assay and in its ability to be reduced to ferroperoxidase, to be converted to oxyferroperoxidase (compound III), and to form peroxidase complexes with hydrogen peroxide (compounds I and II). In contrast to the HRP, the tomato peroxidase fails to catalyze the aerobic oxidation of indole-3-acetic acid in the presence of 2,4-dichlorophenol and manganese. The tomato peroxidase can be resolved into two nonidentical subunits in the presence of dithiothreitol while HRP remains as a single polypeptide chain after such treatment. Dithiothreitol is oxidized in the presence of tomato or horseradish peroxidase with the enzymes accumulating in their oxyferroperoxidase forms during the oxidation reaction. Whereas HRP returns to its free ferric form at the end of the reaction, the tomato enzyme is converted into a form that absorbs at 442 nanometers.

Journal Article↗
PubMed1987

Production and Preliminary Characterization of Monoclonal Antibodies against Cationic Peanut Peroxidase.

Ten monoclonal antibodies (McAbs) have been produced against the cationic peroxidase from peanut suspension cell culture. Eight of these antibodies were found to be of the immunoglobulin (Ig)G(1) subclass and two were of IgA subclass. A combination of competitive enzyme-linked immunosorbent assay, Western blotting analysis, and direct antigen-binding assay revealed that the antibodies are directed against four different epitopes on the cationic peroxidase and the McAbs can be subdivided into four groups. Only group A inhibits peroxidase activity. Group B and D bind equally well to the native and the denatured form of cationic peroxidase, whereas the remaining McAbs react with more or less reduced affinity to the denatured antigen. Group C probably recognizes a conformation-dependent epitope. All the McAbs cross react weakly with the anionic peanut peroxidase, suggesting a structural nonidentity as well as some similarity between these two peroxidase isozymes. Cross reactivities of these McAbs with peroxidases of various plant species were also demonstrated.

Journal Article↗
PubMed1988

Induction of 33-kD and 60-kD Peroxidases during Ethylene-Induced Senescence of Cucumber Cotyledons.

Ethylene enhanced the senescence of cucumber (Cucumis sativus L. cv ;Poinsett 76') cotyledons. The effect of 10 microliters per liter ethylene was inhibited by 1 millimolar silver thiosulfate, an inhibitor of ethylene action. An increase in proteins with molecular weights of 33 to 30 kilodaltons and lower molecular weights (25, 23, 20, 16, 12, and 10 kilodaltons) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after ethylene enhanced senescence. The measurement of DNase and RNase activity in gels indicated that these new proteins were not nucleases. Two proteins from ethylene-treated cotyledons were purified on the basis of their association with a red chromaphore and subsequently were identified as peroxidases. The molecular weights and isoelectric points (pI) of two of these peroxidases were 33 kilodaltons (cationic, pI = 8.9) and 60 kilodaltons (anionic, pI = 4.0). The observation that [(35)S]Na(2)SO(4) was incorporated into these proteins during ethylene-enhanced senescence suggests that these peroxidases represent newly synthesized proteins. Antibodies to the 33-kilodalton peroxidase precipitated two in vitro translation products from RNA isolated from ethylene-treated but not from control cucumber seedlings. This indicates that the increase in 33-kilodalton peroxidase activity represents de novo protein synthesis. Both forms of peroxidase degraded chlorophyll in vitro, which is consistent with the hypothesis that peroxidases have catabolic or scavenging functions in senescent tissues.

Journal Article↗
PubMed1992

Increased Activity of a Cationic Peroxidase Associated with an Incompatible Interaction Between Xanthomonas oryzae pv oryzae and Rice (Oryza sativa).

Rice (Oryza sativa L.) cultivar Cas 209 carries the gene Xa-10 for resistance to race 2 of Xanthomonas oryzae pv oryzae, the bacterial blight pathogen. When seedling leaves of Cas 209 plants were infiltrated with bacterial cell suspensions of strain PXO86(Rif) (race 2, incompatible), total peroxidase activity in extracts from extracellular spaces increased almost threefold between 16 and 24 hours after inoculation. The increase in total peroxidase activity in extracellular extracts was correlated with the appearance of a 43-kilodalton peroxidase isoenzyme with an isoelectric point of 8.6. Increases in the activities of two anionic peroxidase isoenzymes also were associated with the incompatible interaction. Later during the interactions, total peroxidase activities increased in both compatible (cv Cas 209 infiltrated with race 1, PXO61(Sm)) and control (Cas 209 infiltrated with water) treatments, but final activity levels were less than that observed in the incompatible combination. Similarly, the cationic peroxidase was detected in all three treatments by 48 hours after infiltration, but at reduced levels in compatible and water-infiltrated control treatments relative to the incompatible combination. Accumulation of this peroxidase in extracellular spaces thus may play a role in the defense response in cultivar Cas 209.

Journal Article↗
PubMed1980

Immuno-electron microscopical investigations with a new tracer: peroxidase-labeled protein A: application for detection of pemphigus and bullous pemphigoid antibodies.

Peroxidase-labeled Protein A, stable immunoenzyme tracer of high reactivity and comparatively low molecular weight, has been applied in immuno-electron microscopy for detection of bound IgG-type pemphigus and bullous pemphigoid antibodies. Comparing Protein A-peroxidase with peroxidase-labeled immunoglobulins, we obtained similar morphological results in corresponding incubation techniques, but lower nonspecific adsorption of Protein A-peroxidase complexes in tissues. The Protein A-peroxidase molecules showed good tissue penetration abilities. Our rapid one-step incubation procedure led to enhanced preservation of tissue fine structures, without the need of prior tissue fixation. It seems that Protein A-peroxidase is able to replace peroxidase-labeled anti-IgG for immuno-electron microscopical purposes.

Humans↗
PubMed2004

Peroxidase activity associated with suberization processes of the muskmelon (Cucumis melo) rind.

The rind of fruits of muskmelon (Cucumis melo L. var. reticulatus) contains a network of suberized tissue referred to as the 'netting', and peroxidase (EC 1.11.1.7) activity is necessary to the polymerization of the aromatic domain of suberin. Peroxidase activity increased dramatically during the early stages of melon fruit netting, and in fruits exhibiting incomplete netting, peroxidase activity was significantly higher in netted than in non-netted regions of the same fruit. Moreover, analysis of peroxidase activity in three varieties of smooth-rind melons (Cucumis melo var. inodorous) indicated lower levels of the activity in rind samples, taken throughout fruit development, than in rinds of netted varieties. Netting-associated anionic peroxidase (NAPOD) was isolated from the melon rind at an early stage of netting development, partially purified, microsequenced and its cDNA was cloned. It was found to be a single-copy gene within the genome of netted and smooth melon varieties, and highly homologous to other Cucurbitaceous anionic peroxidases. A high transcript level was only detected in the rind of the netted variety. Monitoring the gene expression of netting-associated anionic peroxidase, together with other enzymes involved in the netting will shed light on the molecular control of the suberization processes in the melon rind and in plants in general.

Journal Article↗