• Title/Summary/Keyword: Isoenzymes

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Purification and Characterization of Urushiol Induced Laccase Isoenzyme from Fomitella fraxinea (Urushiol에 의해 유도된 장수버섯 laccase isoenzyme의 정제 및 특성)

  • Choi, Han-Seok;Park, Hyo-Suk;Yeo, Soo-Hwan;Jeong, Seok-Tae;Choi, Ji-Ho;Kim, Myung-Kon
    • The Korean Journal of Mycology
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    • v.38 no.2
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    • pp.152-159
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    • 2010
  • The influence of urushiol, as an allergen on laccase property of Fomitella fraxinea was investigated. The enzyme production was reached to the highest level after 10 days, cultivation and the activity and mycelial biomass were increased by 2.5 and 1.5 folds, respectively, by adding urushiol in the culture medium. In liquid cultures using a Cu Mn-free medium, laccase lactivity was decreased by 3.8-9.2 folds, with similar dry cell weight. Two isoenzymes, were purified using anion exchange, hydrophobic interaction and size-exclusion chromatographies. Both isoenzymes are monomeric proteins, with $M_W$ around 67 kDa(Lac1) and 66 kDa(Lac2), and isoelectric points of 3.67 and 3.81. The optimal conditions for purified isoenzymes were found to be pH 4.5-5.0 and $30-35^{\circ}C$. Activity decreased by the addition of $Fe^{2+}$, $Mg^{2+}$, $Na^+$, and strongly inhibited by EDTA and sodium azide.

Glutathione S-Transferase Expression in Upper Urinary Tract Urothelial Carcinomas: a Taiwan Study

  • Chen, Szu-Han;Wu, Wen-Jeng;Tu, Hung-Pin;Li, Wei-Ming;Huang, Chun-Nung;Li, Ching-Chia;Lin, Hui-Hui;Ke, Hung-Lung
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.11
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    • pp.6475-6479
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    • 2013
  • Objectives: Glutathione S-transferase (GST) isoenzymes play important roles in resistance to cell apoptosis and carcinogenesis. We aimed to establish the relationship between GST expression and the prognosis of upper urinary tract urothelial carcinoma (UTT-UC) in Taiwan. Methods: This study retrospectively reviewed 46 patients with pathologically confirmed UUT-UC at Kaohsiung Medical University Hospital. In each patient, expression of GSTT1 and GSTP1 was compared between urothelial carcinoma and normal urothelial cells by Western blotting. Results: GSTP1 expression in the UUT-UC cells was significantly higher than that in normal urothelial cells (1.6 fold, p<0.001). Expression of GSTT1 was significantly associated with the invasiveness of the carcinoma (p=0.006). Conclusions: In UUT-UC, GSTP1 might be a potential tumor marker, whereas high GSTT1 expression could be used as an indicator of cancer progression. This study is the first to demonstrate potential applications of different GST isoenzymes for biomolecular analysis of UUT-UCs in Taiwan.

Purification and Characterization of Six Fibrinolytic Serine-Proteases from Earthworm Lumbricus rubellus

  • Cho, Il-Hwan;Choi, Eui-Sung;Lim, Hun-Gil;Lee, Hyung-Hoan
    • BMB Reports
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    • v.37 no.2
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    • pp.199-205
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    • 2004
  • The six lumbrokinase fractions (F1 to F6) with fibrinolytic activities were purified from earthworm Lumbricus rubellus lysates using the procedures of autolysis, ammonium sulfate fractionation, and column chromatography. The proteolytic activities on the casein substrate of the six iso-enzymes ranged from 11.3 to 167.5 unit/mg with the rank activity orders of F2 > F1 > F5 > F6 > F3 > F4. The fibrinolytic activities of the six fractions on the fibrin plates ranged from 20.8 to 207.2 unit/mg with rank orders of F6 > F2 > F5 > F3 > F1 > F4. The molecular weights of each iso-enzyme, as estimated by SDS-PAGE, were 24.6 (F1), 26.8 (F2), 28.2 (F3), 25.4 (F4), 33.1 (F5), and 33.0 kDa (F6), respectively. The plasminogen was activated into plasmin by the enzymes. The optimal temperature of the six iso-enzymes was $50^{\circ}C$, and the optimal pH ranged from pH 4-12. The four iso-enzymes (F1-F4) were completely inhibited by PMSF. The two enzymes (F5 and F6) were completely inhibited by aprotinin, TLCK, TPCK, SBTI, LBTI, and leupeptin. The N-terminal amino acid (aa) sequences of the first 20 to 22 residues of each fraction had high homology. All six isoenzymes had identical aa residues 2-3 and 13-15. The N-terminal 21-22 aa sequences of the F2, F3, and F4 isoenzymes were almost the same. The N-terminal aa sequences of F5 and F6 were identical.

Purification and Thermal Inactivation of Two Lipoxygenase Isoenzymes from Potato Tubers (감자 Lipoxygenase isozyme의 분리와 열불활성화)

  • Kim, Young-Myeong;Lee, Chang-Won;Park, Kwan-Hwa
    • Korean Journal of Food Science and Technology
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    • v.19 no.5
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    • pp.397-402
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    • 1987
  • Two lipoxygenases (F-I and F-II) were purified from potato tubers by ammonium sulfate fractionation and ion-exchange column chromatographies. The purified isoenzymes were apparently homogeneous on polyacrylamide gel electrophoresis. Both enzymes showed a similar optimum pH of 5.5-6.0. From thermal inactivation experiments with the purified enzymes in the range of 50 to $65^{\circ}C$, D-values of 13.3 min and 4.3 min at $65^{\circ}C$, and z-values of $11.8^{\circ}C\;and\;10.3^{\circ}C$ were obtained respectively for F-I and F-II. By applying absolute reaction rate equation, thermodynamic parameters wire also determined for the activation part of the inactivation process.

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Study on Productivity and Genotype Structure by Several Enzyme Loci of Silkworm(Bombyx mori L.) Parthenoclones Obtained by Thermal and Combined(Low-High Temperature) Method

  • Vasileva, Jolanda Bogdanova;Staykova, Teodora Atanasova;Tzenov, Panomir Ivanov
    • International Journal of Industrial Entomology and Biomaterials
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    • v.18 no.2
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    • pp.131-134
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    • 2009
  • The purpose of this study was to establish whether there are differences in the productivity of the same silkworm(Bombyx mori L.) parthenoclones, obtained by two different methods-thermal and combined, as well as to study their genotype structure by several enzyme loci. It was established that all individuals of parthenoclones Joana, Joana(${\downarrow}{\uparrow}$), Pohi and Pohi(${\downarrow}{\uparrow}$), are homozygous by the studied esterase and phosphoglucomutase loci, which substantiated the clones' genetic stability. By comparative analysis of some biological and technological properties, it was found that parthenoclone Pohi(${\downarrow}{\uparrow}$) obtained by low-high temperature activation is characterized by higher values of these properties as compared to parthenoclone Pohi obtained by thermal parthenogenesis. Comparing the two methods of inducing ameiotic parthenogenetic development, we would recommend that parthenoclone Joana is sustained by thermal parthenogenesis, and parthenoclone Pohi-by the combined method (low-high temperature).

Modifications of Skim Milk Protein by Meju Protease and Its Effect on Acid Clotting and Digestibility (메주 단백질 가수분해 효소 처리가 탈지 우유 단백질의 응고물 형성 및 소화율에 미치는 영향)

  • 이진실
    • Journal of Nutrition and Health
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    • v.26 no.8
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    • pp.998-1005
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    • 1993
  • This study was attempted to investigate the effects of enzymatic modification of milk protein with Meju protease on its acid clotting and digestibility. The proteases used in this study were isolated from Meju(fermented soybeans) and had specific acticity of 250 units/mg protein at pH 7.0. These proteases were found to be at least 3 different isoenzymes of different pH optima(pH 4.0, 6.0, 10.0). The optimum temperature was 5$0^{\circ}C$. Hydrolyzed skim milk showed 30.5% degree of hydrolysis for 1 hr. and 36.4% degree of hydrolysis for 3.5 hrs. of protease treatment at pH 7.0. Upon acidification to pH 4.0, skim milk produced large and dense coagulum, but the coagulum was getting smaller by protease treatment. Generally, digestability of skim milk at pH 4.0 was lower than pH 2.0. At pH 4.0, native skim milk and control group had problem with hydrolysis of skim milk protein. Among protease treated groups, 1 hour treated skim milk was most effectively hyrolyzed at pH 4.0.

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Purification and characterization of polyphenol oxidase from fresh ginseng

  • Kim, Jae-Joon;Kim, Woo-Yeon
    • Journal of Ginseng Research
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    • v.37 no.1
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    • pp.117-123
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    • 2013
  • Polyphenol oxidase (PPO) was purified from fresh ginseng roots using acetone precipitation, carboxymethyl (CM)-Sepharose chromatography, and phenyl-Sepharose chromatography. Two isoenzymes (PPO 1 and PPO 2) were separated using an ion-exchange column with CM-Sepharose. PPO 1 was purified up to 13.2-fold with a 22.6% yield. PPO 2 bound to CM-Sepharose, eluted with NaCl, and was purified up to 22.5-fold with a 17.4% yield. PPO 2 was further chromatographed on phenyl-Sepharose. The molecular weight of the purified PPO 2 from fresh ginseng was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was about 40 kDa. The optimum temperature and pH were $20^{\circ}C$ and 7.0, respectively, using catechol as a substrate. Pyrogallol showed the highest substrate specificity. The effect of a PPO inhibitor showed that its activity increased slightly in the presence of a low concentration of citric acid. High concentrations of acidic compounds and sulfite agents significantly inhibited purified ginseng PPO 2.

Molecular Markers and Their Application in Mulberry Breeding

  • Vijayan, Kunjupillai
    • International Journal of Industrial Entomology and Biomaterials
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    • v.15 no.2
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    • pp.145-155
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    • 2007
  • Mulberry (Morus spp.) is an economically important tree crop being cultivated in India, China and other sericulturally important countries for its foliage to feed the silk producing insect Bombyx mori L. Genetic improvements of mulberry lag behind to the same in many other economically less important crops due to the complexity of its genetics, the breeding behavior, and the lack of basic information on factors governing important agronomic traits. In this review, the general usage and advantages of different molecular markers including isoenzymes, RFLPs, RAPDs, ISSRs, SSRs, AFLPs and SNPs are described to enlighten their applicability in mulberry genetic improvement programs. Application of DNA markers in germplasm characterization, construction of genetic linkage maps, QTL identification and in marker-assisted selection was also described along with its present status and future prospects.