Cationic Isoperoxidases Research Articles

Purification and Characterization of an Anionic Isoperoxidase from Scented‐Geranium Callus

Secretory anionic isoperoxidase (EC 1.11.1.7), named PA1, was 68‐fold purified from scented‐geranium (Pelargonium graveolense) callus by using ion exchange chromatography and gel filtration. Isoperoxidase PA1 was a glycoprotein with an isoelectric point (pI) of 4.0. The molecular weight of PA1 was approximately 42.5 and 44 kDa, estimated by SDS–PAGE and Sephadex G‐150 gel filtration, respectively. The optimum pH of the enzyme was 5.0 for guaiacol and H2O2, and the K m values for guaiacol and H2O2 were 1.96 and 8.5 mM, respectively. Substrate studies in terms of optimum pHs and K m values with various synthetic and naturally occurring phenolic compounds were performed. In comparison with cationic isoperoxidase, PC3, which has been already characterized, anionic isoperoxidase PA1 had much lower K m values for synthetic phenolic compounds and much higher K m values for naturally occurring phenolic compounds than PC3. Moreover, anionic isoperoxidase PA1 could utilize ferulic acid as a substrate very well, while cationic isoperoxidase PC3 could not utilize ferulic acid as a substrate.

Inhibition of Al-induced root elongation and enhancement of Al-induced peroxidase activity in Al-sensitive and Al-resistant barley cultivars are positively correlated

The quantitative changes in peroxidase activity and composition of anionic and cationic isoperoxidases were investigated in roots of two barley cultivars differing in Al resistance. Root growth of Al-resistant cv. Bavaria was in lesser extent reduced by Al treatment (23% after 24 h Al-treatment), whereas 40% reduction of the root growth was observed in Al-sensitive cv. Alfor. The strong root growth inhibition in Al-sensitive cv. Alfor correlated with a 6-fold enhancement of peroxidase activity by Al treatment. Al-induced enhancement of peroxidase activity was found also in roots of Al-resistant cv. Bavaria, but this increase was only half of the Al-sensitive cv. Alfor. Comparison of peroxidase isoenzyme composition of Al-treated and non-treated roots revealed that activity of at least five anionic and four cationic isoperoxidases was stimulated by Al treatment. Three of anionic isoperoxidases (aPOD2-4) were selectively induced only in the Al-sensitive cv. Alfor. A possible involvement of peroxidases in root-growth inhibition is discussed.

Peroxidase from oranges ( Citrus sinenses (L.) Osbeck

Soluble peroxidase was extracted from oranges (Citrus sinenses (L.) Osbeck). Small amounts of purified anionic and cationic isoperoxidases have been obtained by gel filtration and ion-exchange chromatography. It has been shown that peroxidase activity present in crude extracts of oranges (juice, albedo, and peel) is less stable to heat than the enzymes activity of highly purified individual orange isoperoxidases. For the purified isoperoxidases heat-inactivation is still non-linear. It is suggested that this may be due to micro heterogeneity in covalently bound oligosaccharide residues at the molecular level. The higher activity was detected in the crude extract from the peel, around 3.2 and 7.8 times more than the fractions from the albedo and juice respectively. The crude soluble peroxidase fractions were particularly heat stable. The pI (4.5, 5.2, and 9.0) of the purified isoperoxidase (A1, A3, and C1) from the peel soluble fraction was measured using a surface electrode and the molecular weight (42 kDa, 30 kDa, and 26 kDa) was estimated by gel-filtration.

Effects of heat treatment on chlorophyll degrading enzymes in stored broccoli (Brassica oleracea L.)

Abstract Effects of heat treatment on chlorophyll (Chl) degrading enzyme activities in stored broccoli ( Brassica oleracea L.) were determined. Chl contents of broccoli treated at 50 °C for 1 h or without heat treatment (control) decreased after 4 days of storage at 15 °C, whereas the contents of broccoli treated at 50 °C for 2 h showed almost no change after 4 days of storage at 15 °C. Chlorophyllase activity of broccoli heat-treated for 1 or 2 h decreased during storage before the occurrence of yellowing, while the activity of the control showed a slight decrease on day 4. Chl oxidase activity of broccoli heat-treated for 2 h remained unchanged. Chl degrading peroxidase activity in the control markedly increased after 6 days of storage at 15 °C, but an increase in the activities in broccoli treated at 50 °C, especially for 2 h, was suppressed. Six anionic and two cationic isoperoxidases were detected in broccoli on day 0. One of the isoperoxidases, C 2 (Rf 0.3) cationic isoperoxidase, was involved in Chl degradation and the increase in C 2 level was greatly reduced by treatment at 50 °C for 2 h. These results indicate that heat treatment could reduce Chl degradation due to the suppression of Chl degrading enzyme activities.

Identification and characterisation of Ca 2+-pectate binding peroxidases in Arabidopsis thaliana

The Arabidopsis genome encodes many secretory guaiacol peroxidases (class III plant peroxidases, EC 1.11.1.7). These higher plant enzymes are found either in the vacuole or in the apoplast, where several functions have been attributed to them. Their localisation within the cell wall matrix is most likely important for their activity. In the present work, a gel consisting of polygalacturonate chains cross-linked by Ca2+ and embedded in polyacrylamide was used to separate proteins from Arabidopsis leaves having an affinity for the Ca2+-mediated conformation of pectin. This chromatographic technique selected a small number of cationic isoperoxidases able to bind to Ca2+-pectate but not to Ca2+-alginate, a polyuronate gel similar to Ca2+-pectate. This result suggested that some of the Arabidopsis peroxidases have an affinity for pectin in vivo. Such a property could allow them to be properly distributed within the cell wall network. In addition, eleven cDNAs encoding an Arabidopsis peroxidase were expressed in the baculovirus-insect cell system. The capacity of the resulting recombinant peroxidases to bind Ca2+-pectate and Ca2+-alginate was also assessed. It appeared that 3 of them exhibited a Ca2+-pectate binding activity that was resistant to the action of NaCl. The binding of these recombinant peroxidases to Ca2+-alginate was much weaker than to Ca2+-pectate, confirming the specificity of the interaction with the pectic structure.

Effects of Na 2SO 3 on the activities of antioxidant enzymes in geranium seedlings

This study investigated the effects of Na 2SO 3, which releases SO 2 in apoplastic water, on the growth of geranium seedlings and on the activities of various antioxidant enzymes including peroxidase. Sodium sulfite (Na 2SO 3) addition both inhibited primary root growth and stimulated lateral root growth of the seedlings respectively. In addition, the contents of chlorophyll and Rubisco protein of the seedlings were greatly reduced with Na 2SO 3 treatment. Total peroxidase activities of the seedlings also increased proportionally with the amount of Na 2SO 3 , this presumably correlating with oxidative stress levels. Notably, about an 8-fold enhancement of total peroxidase activity occurred in seedlings treated with 60 nM Na 2SO 3 at pH 4.0. This enhancement of total peroxidase activity was mainly due to the increase of a strong cationic isoperoxidase, strong anionic isoperoxidase and neutral isoperoxidase activities. The strong cationic isoperoxidase from geranium seedlings was found to be the same enzyme as PC3 from geranium callus in terms of its physicochemical and catalytic properties. Moreover, the activities of superoxide dismutase and glutathione reductase were greatly enhanced with Na 2SO 3 treatments at pH 4.0 without significant alteration of catalase activity. These results suggest that Na 2SO 3 exposure, activities the plants defense mechanism against the reactive oxygen species generated.

Purification and characterization of a cationic isoperoxidase from scented-geranium

A strongly cationic isoperoxidase named PC3 was purified to homogeneity from scented-geranium ( Pelargonium graveolens) callus by using DEAE-Sephacel chromatography, CM–cellulose chromatography and Sephacryl S-200 gel filtration, respectively. The enzyme was a glycoprotein with M r of ca. 58 kDa estimated by SDS–PAGE and Sephadex G-150 gel filtration. The pI value of the enzyme was 9.1. Kinetic studies revealed that PC3 had a very low K m value for scopoletin of 0.01 mM and could use ascorbate as a substrate. Interestingly, PC3 could not oxidize ferulic acid as a substrate. Chemical modification of the enzyme showed that PC3 was rapidly inactivated by His, Cys, Trp and Lys-specific reagents. The second order rate constants and reaction orders with respect to these inactivations were determined. Notably, ca. 4–7-fold activity boosting of PC3 occurred by adenine and imidazole when anilino substrates, such as o-dianisidine and o-phenylenediamine were oxidized, whereas this activity boosting did not occur when several phenolic substrates were used.

The regulation of 5′ upstream regions of a Korean radish cationic peroxidase gene by gibberellic acid and abscisic acid

The Korean radish cationic peroxidase promoter (KRPP) and the β-glucuronidase (GUS) gene were fused on the genome of tobacco BY-2 cells via Agrobacterium-mediated transformation method. A fragment of the KRPP comprising nucleotides −471 to +704 relative to the transcriptional initiation site confers constituent expression of GUS in transgenic tobacco BY-2 cells (BYK12). The expressed GUS activity was increased by as much as 40% at 577 nM gibberellic acid (GA 3). This increase was time and dose dependent. The abscisic acid (ABA) from 10 to 100 μM slowly decreased the GUS activity and at 100 μM reduced completely the inductive effect of GA 3 on GUS expression. Thus, GA 3 and ABA have antagonistic effects on the GUS expression mediated by the Korean radish cationic peroxidase promoter in transgenic BYK12 cells. Exogenous GA 3 activated the activity of the Korean radish isoperoxidases and also increased the intensity of two bands of Korean radish cationic isoperoxidases, when hypocotyl portions of the Korean radish seedlings grown in a green house for 10 days were examined using starch gel electrophoresis. However, ABA decreased the intensity of the Korean radish cationic isoperoxidase bands and also reduced the inductive effect of GA 3 in seedlings.

Hydrogen donor specificity of mango isoperoxidases

Purified mango cationic and anionic isoperoxidases catalyse the oxidation of guaiacol, o-dianisidine and 2,2′-azino-di-3-ethyl-benzothiazoline sulphonate (ABTS) at different rates. For an anionic isoperoxidase (A1) o-dianisidine is the most susceptible substrate, whereas for a cationic isoperoxidase (C1) ABTS is oxidised more rapidly. For indoleacetic acid (IAA), mixtures of anionic isoperoxidases catalysed the oxidation at higher rates than cationic isoperoxidases relative to their peroxidase activity towards o-dianisidine. For four purified isoenzymes the greatest rate of oxidation of IAA was found for A1. For dihydroxyfumarate (DHFA), mixtures of cationic isoperoxidases catalysed the oxidation at approximately twice that observed for anionic isoperoxidases per unit of peroxidase activity. Likewise, purified mango cationic isoperoxidases (C1 and C2) oxidised DHFA at a rate three times greater than that for the anionic isoperoxidases of identical peroxidase activity towards o-dianisidine. The mango anionic peroxidases have a greater molecular mass than the cationic isoenzymes. Molecular weights determined by gel filtration of 40 000, 44 000, 22 000 and 27 000 were found for mango isoperoxidases A1, A2, C1 and C2.

Characteristics of six isoperoxidases from Korean radish root

Two cationic isoperoxidases (designated C 1 and C 3) and four anionic isoperoxidases (designated A 1, A 2, A 3n and A 3) from Korean radish ( Raphanus sativus L.) root have been purified to apparent homogeneity, and some of their enzymatic properties were characterized. All six isoperoxidases are glycoproteins composed of a single polypeptide chain. The M r s Of C 1, C 3, A 1 and A 2 were ca 44 000, while anionic isoperoxidase A 3n and A 3 have M r s of 31 000 and 50 000, respectively. Deglycosylated A 2 and C 3 by trifluoromethanesulphonic acid treatment showed M r s of 37 000 and 40 000, respectively, suggesting that the carbohydrate contents for these isoenzymes are 14 and 9%, respectively. Relative amino acid compositions of four anionic isoperoxidases (designated A 1, A 2, A 3n and A 3) and one cationic isoperoxidase C 3 were determined. N-Terminal amino acid sequences were determined for A 1, A 3n and C 3, while A 2 was found to have a blocked amino terminal residue. Kinetic studies with respect to various synthetic and naturally occurring substrates were also investigated.

Peroxidase Activity in the Xerophytic «Cardon» ( Pachycereus pringlei), a Cactaceae of the Sonoran Desert of Mexico

Several isoperoxidase activities (EC 1.11.1.7. donor: H2O2 oxidoreductase) were detected in branch extracts of the cardón cactus (Pachycereus pringlei) of the Sonoran Desert, México. The whole enzyme activity in the crude extract was separated into several anionic and cationic isoenzymes. These isof orms exhibited characteristics similar to those of other plant peroxidases, i.e. i) optimal pH, ii) high activity when using guaiacol as hydrogen donor, iii) lower Vmax for DOPA and iv) isoperoxidases were inactivated at high concentrations of the substrate hydrogen peroxide. However, cardón peroxidases were denatured at 70 °C. In FPLC Mono S, the main active fractions were separated into an anionic and two cationic isoperoxidases. When using isoelectric focusing, the anionic isoperoxidase was separated into two bands with slightly different isoelectric points. The partial purification of the crude extract using ammonium sulfate fractionation showed a highly atypical behavior, yielding a purification factor of 12 at 40 – 90% saturation range

The thermostability of purified mango isoperoxidases

Small amounts of purified mango (var. Chaunsa) anionic and cationic isoperoxidases have been obtained by ion-exchange chromatography. It has been shown that peroxidase activity present in crude extracts of mango pulp is less stable to heat than the enzymic activity of highly purified individual mango isoperoxidases. For the purified isoperoxidases heat-inactivation is still nonlinear. It is suggested that this may be due to microheterogeneity in covalently bound oligosaccharide residues at the molecular level. Isoperoxidase activity did not regenerate after heat treatment of crude mango extracts or purified isoenzymes.

Characterization of the Peroxidase System in Winter Rye Seedlings:Compartmentation and Dependence on Leaf Development and Hydrogen Donors Used

Summary The peroxidases of winter rye ( Secale cereale L.) seedlings were characterized by means of pH optimum, compartmentation and changes in activity and isoperoxidase pattern during primary leaf development. Peroxidase activity was measured with 9 different hydrogen donors in order to find donor-associated peculiarities. There are a number of parameters that depend on hydrogen donors: i) pH optima; ii) time course; iii) distribution along the leaf; iv) cellular compartmentation and v) isoperoxidase pattern. Gradual changes were found for hydrogen donors according to the character of compartmentation and distribution of peroxidase activity during leaf development, in a sequence dianisidine — guaiacol — catechol — vanillin — ascorbate. In contrast to dianisidine peroxidase activity, which was at a high level in the extracellular fraction of young tissues and sharply increased in older ones, ascorbate peroxidase activity was localized only in protoplasts of juvenile meristematic cells at the basis of the growing leaf. Extraction of tissues with a low ionic strength buffer (0.1 M Na-phosphate, pH 7.8) liberated the main portion (86–91 %) of total peroxidase activity. Both anionic and cationic isoperoxidases were localized intracellularly and extracellularly. The increase in total soluble peroxidase activity during leaf development was associated with the increase in activity of all the presented isoforms, with the exception of A3 and A4, but not with an appearance of new ones.

Near-Isogenic Wheat Suspension Cultures: Establishment, Elicitor Induced Peroxidase Activity and Potential Use in the Study of Host/Pathogen-Interactions

Summary Callus and suspension cultures were established from two near-isogenic lines of Triticum aestivum, cultivar Prelude, resistant and susceptible to stem rust. The cultures were characterized with respect to growth kinetics, pH, nutrient uptake, protein and peroxidase secretion into the culture medium. Among other differences, higher extracellular peroxidase activities were observed in the resistant line. These differences in peroxidase activity were even more obvious when the suspension cultures were treated with elicitor-active materials that included Pgt-elicitor isolated from stem rust germ tubes. Treatment with Pgtelicitor resulted in a significant increase in extracellular peroxidase activity in suspension cultured cells originating from the resistant wheat line, while the culture originating from the susceptible wheat line was not affected. Remarkably, chitinoligomers, chitosan and DEAE-dextran stimulated peroxidase activity in both cell cultures. Further investigations using gel electrophoresis revealed that in addition to some anionic isoperoxidases mainly cationic isoperoxidases respond to elicitors. The results suggest that the cell cultures provide a suitable system to investigate biochemical and molecular biological aspects of the wheat/rust-interaction, including the mode of elicitor action.

Changes in chloroplast peroxidase activities in relation to chlorophyll loss in barley leaf segments

Total peroxidase activity increased during senescence of excised barley (Hordeum vulgare L. cv. Kashimamugi) leaves. Kinetin treatment furter increased total peroxidase activity but repressed chlorophyll degradation in excised barley leaves. When isoperoxidases were extracted from barley leaf segments. 4 cationic and 4 anionic isozymes were found in polyacrylamide gel electrophorests during leaf senescence. The chloroplasts contained only two cationic isoperoxidase activities. One (designated C4) was repressed by kinetin. and the other (C3) was increased by kinetin. Glucosamine, which also repressed the degradation of chlorophyll, completely repressed C4 activity but did not affect C3 activity. The induction with senescence, and the repression with kinetin and glucosamine, suggest chat chloroplast isoperoxidase C4 may function as a chlorophyll‐degrading enzyme during barley leaf senescence.

Indole‐3‐acetic acid oxidase and syringaldazine oxidase activities of peroxidase isozymes in soybean root nodules

Many isoperoxidases with indole‐3‐acetic acid oxidase (IAA) and syringaldazine oxidase activities were detected by polyacrylamide gel electrophoresis in soybean root nodules [Glycine max (L.) Merrill, cv. Asgrow], detached at the onset of flowering. The kinetics of the two activities were studied with some of the isoperoxidases partially purified by ion exchange chromatography. IAA oxidase activity of the cationic isoforms showed a sigmoidal kinetic behaviour and a higher substrate affinity than the anionic ones, whereas typical saturation kinetics were found with an anionic fraction that contained leghemoglobins. So, nodule IAA oxidase activity may mainly be displayed by the cationic isoforms. These cationic isoperoxidases had high affinity towards syringaldazine and they also may be associated with cell wall rigidification.

Production and characterisation of monoclonal antibodies to a cationic peroxidase from spinach leaves

Five different murine monoclonal antibodies specific for a cationic isoperoxidase (C1) prepared from spinach leaves have been produced and characterised. Immunoassays showed that they have different affinities for the purified C1 and that they recognise different epitopes of the peroxidase molecule. Immunoaffinity chromatography, using McAbs-Sepharose columns, has shown that some of the eight isoperoxidases present in a leaf extract share at least one similar epitope, recognised by one McAb. An immunoassay was developed that permits quantitation of purified C1. Two McAbs seem strictly specific for C1, but none of the five McAbs binds proteins other than peroxidases, suggesting that they could all be directed against the peptidic part of the glycoprotein. One McAb specific for C1 also strongly inhibits its enzyme activity, but has no effect on the activity of the other isoperoxidases. Kinetic studies have shown that the inhibition occurs very rapidly after the addition of the McAb, either in classical peroxidase reaction, or during the peroxidase-mediated oxidation of NADH.

4-Coumarate:coenzyme A ligase and isoperoxidase expression in Zinnia mesophyll cells induced to differentiate into tracheary elements.

When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.

Isoperoxidase pattern and internode length genotype in Pisum

Inbred Pisum sativum lines of known constitution for the intemode length genes Le, La and Cry, and representing four height phenotypes, were grown to the 7-intemode stage in the light. Six cationic isoperoxidases, making up ca. 90% of the activity of stem extracts, were resolved by concave gradient elution from Dowex 50 columns and shown to run as single peroxidase bands on starch gel electrophoresis. They were all able to oxidise IAA in the presence of 2,4-dichlorophenol, but fell into two groups with widely differing IAA oxidase/peroxidase ratios. The isoperoxidase patterns were independent of both genotype and phenotype for internode length, thus making it unlikely that these loci exert their effect on internode extension via control of synthesis of a particular isoperoxidase. Amongst the lines screened polymorphism was detected involving two of the isoperoxidases, and limited F 2 data suggest that these two variants fire determined by alleles of a single gene. Isoperoxidase patterns of stem extracts of 6 other Pisum species did not differ significantly from the two found in P. sativum.