• Applied Biological Chemistry
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• 제43권2호
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• pp.100-105
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• 2000

한국산 무(Raphanus sativus L.)로부터 황산암모늄 분별침전과 CM-cellulose ion exchange chromatography를 사용하여 anionic peroxidase(POD)를 분리하였다. 분리된 한국산 무 anionic POD를 비색효소로, o-tolidine 혹은 4-aminoantipyrine/diethylaniline(4AA/DEA)을 비색기질로 사용하여 630nm에서의 흡광도를 측정함으로써 당 정량반응을 실시하였다. 한국산 무 anionic POD는 비색기질로 4AA/DEA을 사용한 경우 o-tolidine을 사용했을 때 보다 더 높은 비색 반응성을 나타내었다. Glutosr의 농도증가에 따른 검정곡선을 작성하였을 때 o-tolidine과 4AA/DEA을 사용한 경우 상관계수가 각각 r=0.9983과 r=0.9963로서 두 가지 방법의 검정곡선이 모두 좋은 직선성을 보여 주었다. 한국산 무 anionic POD와 horseradish POD의 기질산화에 대한 반응성을 비교하기 위하여 o-dianisidine과 guaiacol Km값을 조사한 결과, 한국산 무 anionic POD가 horseradish POD보다 o-dianisidine에 대해 약 40배, guaiacol 대하여 약 2배의 높은 기질 반응성을 나타내었다. 이러한 결과들은 한국산 무 anionic POD를 비색효소로 사용하여 검출감도가 높은 당 정량용 진단시약을 개발할 수 없음을 보여준다.

• 식물조직배양학회지
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• 제25권4호
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• pp.277-282
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• 1998

장미(Rosa sp. cv Paul's Scarlet) 현탁 배양체의 배지에서는 정상적인 성장 조건하에서 cationic peroxidase (POD)만 관찰되었으나, 효모 세포벽으로부터 추출한 숙주 특이성이 낮은 elicitor를 10 mg glucan/L의 농도로 투여하여 24시간 정도 배양하였을 때 기존의 POD와 뚜렷하게 구분되는 anionic POD가 유도됨을 관찰하였다. Elicitor에 의해 유도되는 prominent한 anionic POD (pI 6.1)와 대조구로서 pI 8.4의 cationic isozyme을 분리한 후 효소학적 성질을 비교하였다. Ammonium sulfate농축, ion exchange chromatography, gel filtration chroma tography를 통하여 약 200배로 농축된 두 효소를 분리하였으며, 이 두 효소의 몇몇 기질에 대한 kinetic parameter들은 매우 의미있는 차이를 보여 주었다. 그 중에서 ferulic acid에 대한 Km은 anionic POD의 경우 4.6 mM, cationic POD는 1.38 mM로 측정되었으며, $\textrm{H}_2\textrm{O}_2$에 대한 Km은 각각 anionic POD가 0.72 mM, cationic isozyme이 0.45 mM로 나타났다. 특히 NADH 산화 반응의 활성은 anionic POD가 cationic POD에 비하여 약 2배 높은 것으로 측정되었다. 0.1 mM coniferyl alcohol의 첨가에 의한 NADH 산화 반응의 억제는 anionic POD fraction에서 약 60% 정도 일어난 반면에 cationic POD fraction에서는 약 15% 정도 일어났다.

• KSBB Journal
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• 제13권5호
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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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• 제39권2호
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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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• 제20권5호
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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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• 제28권3호
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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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• 제31권6호
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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.

• 식물조직배양학회지
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• 제26권4호
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• pp.265-269
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• 1999

고구마 산성 peroxidase (POD) (swpal) 또는 중성 POD(swpnl)을 도입한 형질전환 담배에 다양한 선량의 감마선을 조사한 후 30일에 항산화효소 활성과 식물생장에 미치는 영향을 조사하였다. 감마선은 조사선량에 비례하여 형질전환 식물체와 정상식물에 관계없이 모든 식물체의 생장을 크게 억제시켰다. 50-70 Gy 처리에서 식물체의 신장이 크게 억제되었고 새로운 잎이 발생되지 못하였다. 10-50 Gy의 조사선량에서는 POD, superoxide dismutase, catalase 활성에 큰 변화를 주지 않았다. 이러한 결과는 고구마 POD는 감마선 조사에 의해 유도되는 산화적인 스트레스에 대한 보호효과에 관여하지 않음이 시사된다.

• Journal of Microbiology and Biotechnology
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• 제19권7호
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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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• 제31권1호
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• pp.83-88
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• 2004

스트레스 관련 화합물 및 병원균을 고구마 (Ipomoea batatas)잎에 처리하여 고구마 배양세포에서 분리한 산성 퍼옥시다제 swpa2 유전자의 발현을 RT-PCR로 분석하였다. Swpa2유전자는 건강한 식물체 조직에서는 발현되지 않았으나 각종 스트레스에 의해 발현이 유도되었다. Swpa2는 과산화수소 (440mM)처리에 의하여 강하게 발현이 유도되었고 NaCl (100mM), ABA (0.1mM), methyl jasmonate (MeJA, 0.1mM) 처리에 의하여도 발현이 유도되었다. 그러나 salicylic acid (SA)처리에 의해서는 발현이 유도되지 않아, 이 유전자는 SA와 MeJA의 신호전달에는 서로 다르게 관여함이 시사되었다. 또한 swpa2유전자는 무름병을 유발하는 박테리아(Pectobacterium chrysanthemii)의 감염에 의하여 강하게 발현되므로, 이 POD는 병저항성에 관여함을 알 수 있었다. 따라서 swpa2유전자는 각종 비생물학적 스트레스 뿐 아니라 생물학적 스트레스에 수반되는 산화 스트레스를 극복하는데 관여함이 강하게 제시되었다.