• Applied Biological Chemistry
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• v.43 no.2
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• pp.100-105
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• 2000

Anionic peroxidases (POD) were isolated from Korean radish (Raphanus sativus L.) root by using fractionation with $(NH_4)_2SO_4$ and CM-cellulose ion exchange chromatography and used as the colorimetric enzyme for glucose determination. The chromogen used in this work was o-tolidine or 4-aminoantipyrine/diethylaniline (4AA/DEA) and the colored products were measured at 630 nm. Korean radish anionic POD showed much better colorimetric reaction of glucose determination with 4AA/DEA than with o-tolidine. The r values of calibration curve for glucose determination by o-tolidine and 4AA/DEA were 0.9983 and 0.9963, respectively. In order to compare the reactivity for substrate oxidation by Korean radish POD and horseradish POD, the Km values against o-dianisidine and guaiacol were measured. Korean radish POD had about 40 fold higher affinity for o-dianisidine and 2 fold higher affinity for guaiacol as revealed by Km values. These results showed that Korean radish POD could be developed as the colorimetric diagnosis reagent for glucose determination with high sensitivity.

• Korean Journal of Plant Tissue Culture
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• v.25 no.4
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• pp.277-282
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• 1998

Whereas cationic extracellular peroxidases (PODs) were observed in the suspension cultures of rose (Rosa sp. L. cv Pual's scarlet) grown under normal conditions, new anionic isozymes were induced within 24 hr by the treatment of low host-specific elicitor (10 mg glucan/L media) prepared from yeast cell wall. Prominent anionic (pI 6.1) and cationic POD (pI 8.4) were purified and characterized to understand the physiological role of the enzymes. Both enzymes were purified (ca.200 fold) by the ammonium sulfate precipitation, ion exchange chromate-graphy and gel filtration chromatography. The Km values of the purified anionic POD for ferulic acid and $\textrm{H}_2\textrm{O}_2$ were 4.64 mM and 0.72 mM, whereas those of the cationic POD were 1.38 mM and 0.48 mM, respetively. The activity of the anionic POD as NADH oxidase was twice higher than that of cationic POD. The NADH oxidation in the anionic POD fraction was inhibited by 60% on the addition of 0.1 mM coniferyl alcohol, while that in the cationic fraction was inhibited by 15%.

• KSBB Journal
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• v.13 no.5
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• pp.599-605
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• 1998

Soybean peroxidase was extracted from soybean hulls and purified by ammonium sulfate precipitations (25% and 75% saturation), pl fractionation, and anionic exchange and gel filtration chromatographies (DEAE-Sephadex A-50 and Superose 12). Modlecular weight and pl value were estimated to be ca. 45 kD and 4.2, respectively. Purified soybean peroxidase had an RZ value of 0.43. Compared with horseradish peroxidase, it showed superior thermal and pH stability. Assuming the first-order kinetics, the thermal deactivation rate constant of soybean peroxidase at 80$^{\circ}C$ was about 8 times lower than that of horseradish peroxidase. Deactivation energy was calculated to be 69.3 kcal/mol. Soybean peroxidase showed about 10% higher H2O2 degradation capacity than horseradish peroxidase. Exploiting these advantages, the soybean peroxidase purified from the domestic soybean hull is expected to replace horseradish peroxidase in various applications.

• Journal of Plant Biology
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• v.39 no.2
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• pp.99-105
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• 1996

Immunocytochemistry utilizes the specificity of the antigen-antibody reaction to localize specific antigens in cells or cellular organelles. Here we report the use of monoclonal antibodies, in conjunction with gold-labeled second antibodies to study the ultrastructural localization and tissue distribution of the Mr 98, 000 anionic peroxidase and other wall antigens. The antibody specific for this wall peroxidase, mWP3, labeled mainly the cell wall area. At the tissue level, the Mr 98, 000 peroxidase is located predominantly in the leaf mesophyll, internal coleoptile and sieve elements, but not in the root, as assayed with these procedures. The coleoptile walls were less heavily stained than the walls of leaf mesophyll cells. At the subcellular level, it is localized mainly in intercellular regions of the cell walls. A similar staining pattern was revealed by mWP19, one of anti-$\beta$ glucosidase antibody, though it looked less heavily stained than one with mWP3. In order to serve as a control wall staining using IgM monoclonal antibodies, mWP18 was used. Most of the label is localized over wall regions of cells of the young leaf mesophyll and coleoptile.

• The Korean Journal of Ecology
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• v.20 no.5
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• pp.315-321
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• 1997

Peroxidase activities and isozyme patte군 of the pine needles (Pinus densiflora) were examined and compared in the coastal regions of Anmyum-Do(Choongnam, Taean-Gun) and inland regions of Shinchang-Myun(Choongnam, Asan-City). The pine needle peroxidase from Anmyum-Do showed approximately three times higher specfic activity than Shinchang pine needle peroxidase. The pine needle extracts of Anmyun-Do and Shinchang contained three anionic isoperoxidases, named A1, A2 and A3, when subjected to starch gel electrophoresis at pH 7.0. Cjationic isoperoxidases could not be found in both extracts., However, there existed unique isoperoxidase An only from the extracts of Anmyun-Do pine needles under the salinary environment. Moreover, the specific activities of catalase and glucose-6-phosphate dehydrogenase from Anmyun-Do, known for the inducible enzymes under the stress condition, were about 1.8 times higher than those of Shinchang pine needles. However, the specific activities of other enzymes did not show great differences between the two regions. Considering the above results of the higher specific activity of peroxidase and the unique expression of isoperoxidase An, pine needle peroxidase might involve in the defence mechanism against the salinary stress of Anmyun-Do.

• Applied Microscopy
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• v.28 no.3
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• pp.399-414
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• 1998

Glandular trichomes present on the leaf surface of Drosera capensis were examined using transmission electron microscopy. A large number of stalked glands exist on the adaxial surfaces of the leaf blade. The secretory head is composed of two layers of secretory cells, one layer of middle cells, and the inner tracheids. The secretory cells contain rough endoplasmic reticulum, mitochondria, plastids, Golgi apparatus, and vacuoles. The secretory cells show prominent cell wall ingrowth, and thick cuticle restricted on the subcuticular wall. Frequently, the cuticle has some pores, canal-like structures, showing electron -dense granules being penetrated through them. Ultrastructural localization using diaminobenzidine showed the electron-dense deposits in the vacuole. No peroxidase activity was seen in the cell wall and cytolasm. The activity of peroxidase (POX) isozymes in Drosera which isoelectric point (pI) is 3.6 and some anionic POX isozymes which pIs are laid between 3.6 and 4.6 were especially increased according to the development and the formation of glandular trichomes. Also, the activity of some POX isozymes which isoelectric points are laid between 4.6 and 5.1 were increased in the regions of leaves which has trichomes.

• BMB Reports
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• v.31 no.6
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• pp.548-553
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• 1998

A minor anionic isoperoxidase named $A_{3n}$, was isolated from Korean radish (Raphanus sativus L.) root. Purification of the enzyme was accomplished by CMcellulose chromatography, DEAE-Sephacel chromatography, and Sephadex G-75 gel filtration. The enzyme was a glycoprotein with molecular weight of approximately 31,000 as determined by SDS-PAGE and 33,000 by Sepadex G-150 gel filtration, which is by far the smallest among the reported isoperoxidases. The pI value was 3.5. The optimum pH of the enzyme was 6.5 for guaiacol and $H_2O_2$, and the $K_m$ values for guaiacol and $H_2O_2$ were 13.3 mM and 1.5 mM, respectively. Kinetic studies with various substrates revealed that only A3n, unlike other isoperoxidases from radish, did not use scopoletin as a substrate and had very low $K_m$ value of 0.25 mM for ferolic acid among naturally occurring phenolic substrates.

• Korean Journal of Plant Tissue Culture
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• v.26 no.4
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• pp.265-269
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• 1999

Transgenic tobacco plants expressing either a sweet potato anionic peroxidase (POD) (swpal) or neutral POD (swpnl) were irradiated by gamma radiation, and the gamma radiation-induced biochemical changes in antioxidant enzymes and plant growth inhibition were investigated at 30 days after treatment. Gamma radiation significantly inhibited the growth of all plants regardless of transgenic or nontransformed plants, showing a dose-dependent inhibition. In high dosage of 50 and 70 Gy, plant heights were severely retarded and new leaves does not emerged. No significant changes in antioxidant enzymes such as POD, superoxide dismutase and catalase were observed in all plants regardless of irradiation dosages ranging from 10 to 50 Gy. These results suggest that sweet potato PODs may be not involved in the protection against the oxidative stress induced by gamma radiation.

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.713-717
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• 2009

The dependence of the catalytic properties of horseradish peroxidase on the structural changes of ionic liquids was investigated with two water-miscible ionic liquids, N-butyl-3methypyridinium tetraftuoroborate ([$BMP_y$][$BF_4$]) and 1-butyl-3-methylimidazolium methylsulfate ([BMIM][$MeSO_4$]), each of which shares an anion ($BF_4^-$) or a cation ($BMIM^+$) with 1-butyl-3-methylimidazolium tetraftuoroborate ([BMIM][$BF_4$]), respectively. The oxidation of guaiacol (2-methoxyphenol) with $H_2O_2$was used as a model reaction. In order to minimize the effect of solution viscosity on the kinetic constants of the enzymatic catalysis, the enzymatic reactions for the kinetic study were performed in water-ionic liquid mixtures containing 25% (v/v) ionic liquid at maximum. Similarly to the previously reported results for [BMIM][$BF_4$], as the concentration of [$BMP_y$][$BF_4$] increased, the $K_m$value increased with a decrease in the $k_{cat}$value: the $K_m$value increased markedly from 2.8 mM in 100% water to 12.6 mM in 25% (v/v) ionic liquid, indicating that ionic liquid significantly weakens the binding affinity of guaiacol to the enzyme. On the contrary, [BMIM][$MeSO_4$] decreased the Km value to 1.4 mM in 25% (v/v) ionic liquid. [BMIM][$MeSO_4$] also decreased $k_{cat}$more than 3-folds [from 13.8 $s^{-1}$in 100% water to 4.1 $s^{-1}$in 25% (v/v) ionic liquid]. These results indicate that the ionic liquids interact with the enzyme at the molecular level as well as at a macroscopic thermodynamic scale. Specifically, the anionic component of the ionic liquids influenced the catalysis of horseradish peroxidase in different ways.

• Journal of Plant Biotechnology
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• v.31 no.1
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• pp.83-88
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• 2004

Expression of an anionic peroxidase swpa2 gene isolated from cultured cells of sweetpotato (Ipomoea batatas) was investigated under various stress conditions by RT-PCR. The swpa2 gene was not expressed in any tissues of intact sweetpotato plant grown at the normal condition. The expression of this gene was strongly induced in leaf tissue by treatment of $H_2O$$_2$ (440mM). Treatment of NaCl (100mM), ABA (0.1mM) and methyl jasmonate(MeJA, 0.1mM) also induced the expression of swpa2 gene. Interestingly, salicylic acid (SA, 0.1 mM) did not induce the expression of swpa2 gene, indicating that anionic swpa2 POD is differently involved in SA and MeJA signaling pathways. In addition, swpa2 gene was strongly induced in sweetpoato leaf tissues infected with Pectobacterium chrysanthemi, indicating that swpa2 is involved in defense related to the pathogenesis of P. chrysanthemi in sweetpotato plants. These results strongly suggest that swpa2 gene is involved in overcoming oxidative stresses caused by both abiotic and biotic stress.