• Title/Summary/Keyword: triton X-100

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Comparative Study of Enzyme Activity and Stability of Bovine and Human Plasmins in Electrophoretic Reagents, β-mercaptoethanol, DTT, SDS, Triton X-100, and Urea

  • Choi, Nack-Shick;Hahm, Jeung-Ho;Maeng, Pil-Jae;Kim, Seung-Ho
    • BMB Reports
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    • v.38 no.2
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    • pp.177-181
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    • 2005
  • Effects of common electrophoretic reagents, reducing agents ($\beta$-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.

Enzymatic Conversion of Pyruvic Acid to Tryptophan tinted to Pyruvic Acid-Producing Microorganism (Pyruvic Acid 생산 미생물과 연결된 Pyruvic Acid의 Tryptophan으로의 효소적 전환)

  • 정남현;방원기
    • Microbiology and Biotechnology Letters
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    • v.15 no.5
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    • pp.334-339
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    • 1987
  • Enzymatic conversion of pyruvic acid produced by microorganism to tryptophan was investigated. A luminescent bacteria. Beneckea sp., was used for the production of pyruvic acid. As a source of tryptophanase which synthesizes tryptophan from pyruvic acid, indole and ammonia, whole cells of Enterobacter aerogenes ATCC 10031 were used directly in the reaction mixture. To increase the production of tryptophan, nonionic detergents and nonaqeous organic solvents were used ms reserviors of indole in the reaction mixture. In the case of nonionic detergents, TritonX-100 was very effective. When 1.5% of Triton X-100 was used, 7.7g/$\ell$ of tryptophan was produced at 37$^{\circ}C$ for 48hr. In the case of nonaqueous solvents, 8.7g/$\ell$ of tryptophan was produced at 37$^{\circ}C$ for 48 hr, when 10% of benzene was used. This amount of tryptophan corresponds to conversion of 48% of Indole and 36% of pyruvic acid, respectively.

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Application of in-situ Flushing to the Soil Contaminated by Organic Compounds (유기물질에 의해 오염된 토양에 대한 in-situ세척기법의 적용성 연구)

  • 최상일;류두현;김형수
    • Journal of Korea Soil Environment Society
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    • v.1 no.2
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    • pp.61-72
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    • 1996
  • A series of batch and lab-scale continuous tests were conducted to optimize the design parameters for the full-scale in-situ soil flushing experiments. The cleaning abilities of the surfactant solutions of Tween 80, Triton X-100 and SDS were compared for the soil artificially contaminated by hydrophobic organic contaminants: n-dodecane, naphthalene and anthracene. Tween 80 and Triton X-100 were shown to be efficient for n-dodecane. SDS and Tween 80 were shown to be efficient for naphthalene and anthracene. At the end of each column test, the sorbed amount of surfactant to soil was also measured. Tween 80 was found to be the least adsorbed surfactant to soil. The flushing ability at flowrate of 7 ml/min, was hampered comparing to flowrate of 3 and 5 ml/min. Initial pH of the soil did not significantly affect the flushing efficiencies. Tween 80 was determined as the most harmless surfactant for the Gram(+) and Gram(-) bacteria.

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Prolonged Expression of Exogenous GFP Gene in the Porcine Embryos generated by Intracytoplasmic Sperm Injection-Mediated Gene Transfer

  • Chung, Hak-Jae;Son, NaRae;Han, Joo-Hee;Park, Chun-Gyu;Kim, Kyung-Woon;Park, Mi-Ryung;Hwang, In-Sul;Park, Jin-Ki;Im, Gi-Sun
    • Journal of Embryo Transfer
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    • v.30 no.3
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    • pp.225-228
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    • 2015
  • Understanding the behavior of transgenes introduced into oocyte or embryos is essential for evaluating the methodologies for transgenic animal production. To date, many studies have reported the production of transgenic pig embryos with, however, low efficiency in environment of blastocyst production. The aim of present study was to determine the expression and duration of transgene transferred by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of GFP and then for the transmission of the transgene. Briefly, fresh spermatozoa were bound to exogenous DNA after treatment by Triton X-100 and Lipofectin. When ICSI-MGT was performed using sperm heads with tails removed, the yield of blastocyst (25.3%), treated with Lipofectin (18.8%) and Triton X-100 (19.2%) were observed. Treatments of Lipofectin or Triton X-100 did not further improve the rates of blastocysts. Moreover, the apoptosis rates of embryos were obtained from the control and LIpofectin groups (8.7%, 9.7%, respectively), but were significantly higher in the Triton X-100 group (13.0%). Our results demonstrated that ICSI-MGT caused minimal damage to oocytes that could develop to full term. Moreover, the embryos derived by ICSI-MGT have shown prolonged exogenous DNA expression during preimplantation stage in vivo. However, more efforts will be required to improve the procedures of both sperm treatments cause of high frequency of mosaicisms.

Purification and Characterization of Heat-Tolerant Protease Produced by Bacillus polyfermenticus SCD

  • Choi, Gooi Hun;Jo, Mi Na;Kim, Jin-Man;Kim, Cheon-Jei;Kim, Kee-Tae;Paik, Hyun-Dong
    • Journal of Microbiology and Biotechnology
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    • v.23 no.11
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    • pp.1554-1559
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    • 2013
  • A protease produced by Bacillus polyfermenticus SCD was purified and characterized as a new detergent material. The protease was purified from supernatant produced by B. polyfermenticus SCD, by ammonium sulfate precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, and finally gel filtration chromatography on Sephadex G-50. The molecular mass of this enzyme was 44 kDa based on SDS-PAGE. The optimum temperature and pH were $50^{\circ}C$ and pH 8.0. The ranges of its stability to the pH and temperature were 7.0 to 9.0 and under $40^{\circ}C$, respectively. The enzyme was highly stable in the presence of the surfactants like Triton X-100 (0.1%), showing a 2-fold increase in its proteolytic activity. However, the enzyme was slightly inhibited by the chelating agent EDTA (1 mM). The enzyme has a maximum activity at $50^{\circ}C$ and the activity can be increased by surfactants such as Triton X-100 and Tween 80.

Effect of surfactants on reductive degradation of Endosurfan I and II by ZVM (영가금속에 의한 Endosulfan I과 II의 환원분해에 미치는 계면활성제의 영향)

  • 김진영;김영훈;신원식;전영웅;송동익;최상준
    • Proceedings of the Korean Society of Soil and Groundwater Environment Conference
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    • 2002.09a
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    • pp.187-190
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    • 2002
  • Reductive dechlorination of endosulfans was studied with zero valent metals (ZVMs) and bimetals in aqueous batch reactors. The effect of surfactants was evaluated. Endosulfan was successfully dechlorinated with zero valent iron. However, a bimetal, palladium coated iron (Pd/Fe) showed a highly enhanced reactivity for both endosulfan I and II indicating palladium act as a dechlorination catalyst on the iron. The effect of surfactants on degradation with ZVM has been very controvertible. Variable concentration of a nonionic surfactant, Triton X-100 and an anionic surfactant, SDS were added into the reactor with ZVM. The reaction rates of endosulfan were increased with both surfactants. In the case of Triton X-100, the reaction rate was increased with the increasing surfactant concentration up to 400 mg/L. Addition of small amount of surfactant under the CMC, the reaction rate was increased. However, the enhancing effect was diminished when a higher concentration of surfactant (1,000 mg/L) was used. Current study implicate that the surfactant adsorbed on the metal surface might increase the surface concentration of endosulfan resulting in the increased reaction rate. However, partitioning of endosulfan into the micelle formed at the high concentration of surfactant diminish the enhancing effect by reducing the contact chance between target compound and the metal surface.

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Mechanism of Cadmium Accumulation into the Cell of Cadmium-Ion Tolerant Yeast (카드뮴 내성 효모의 세포내 카드뮴 축적 기작)

  • 유대식;송형익;정기택
    • Microbiology and Biotechnology Letters
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    • v.18 no.3
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    • pp.233-238
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    • 1990
  • The mechanism of intracellular accumulation of cadmium in a cadmium-ion tolerant yeast, Hansenula ammala B-7, which is an extreme cadmium tolerant strain and has the ability to take up a large amount of cadmium was investigated. The amounts of cadmium taken up by the scalded yeast cells were 2 to 3 times more than the value of the living cells. The living Hansenula anomala B-7 cells adsorbed 74% of cadmium taken up onto the other layer of the cells and 26% of it accumulated inside the cells. But the scalded cells adsorbed 98.3% of cadmium taken up and accumulated 1.7% of it inside the cells. A cadmium uptake and its accumulation were accelerated up to 162.3% and 275.4% by Triton X-100 in the living cells, respectively. Whereas in the scalded cell cadmium uptake was not affected by Triton X-100. Furthermore the cadmium uptake and its accumulation were strongly inhibited by metabolic inhibitors like 2,4-dinitrophenol, sodium azide and potassium cyanide in the living cells, but in the scalded cells cadmium uptake was not affected by metabolic inhibitors. These results suggested that the intracellular accumulation of cadmium by the cadmium-tolerant Hansenula anomala B-7 cells was apparently dependent of biological activity, and also gave evidence of the existance of energy-dependent system.

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HQNO-sensitive NADH:Quinone Oxidoreductase of Bacillus cereus KCTC 3674

  • Kang, Ji-Won;Kim, Young-Jae
    • BMB Reports
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    • v.40 no.1
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    • pp.53-57
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    • 2007
  • The enzymatic properties of NADH:quinone oxidoreductase were examined in Triton X-100 extracts of Bacillus cereus membranes by using the artificial electron acceptors ubiquinone-1 and menadione. Membranes were prepared from B. cereus KCTC 3674 grown aerobically on a complex medium and oxidized with NADH exclusively, whereas deamino-NADH was determined to be poorly oxidized. The NADH oxidase activity was lost completely by solubilization of the membranes with Triton X-100. However, by using the artificial electron acceptors ubiquinone-1 and menadione, NADH oxidation could be observed. The activities of NADH:ubiquinone-1 and NADH:menadione oxidoreductase were enhanced approximately 8-fold and 4-fold, respectively, from the Triton X-100 extracted membranes. The maximum activity of FAD-dependent NADH:ubiquinone-1 oxidoreductase was obtained at about pH 6.0 in the presence of 0.1M NaCl, while the maximum activity of FAD-dependent NADH:menadione oxidoreductase was obtained at about pH 8.0 in the presence of 0.1M NaCl. The activities of the NADH:ubiquinone-1 and NADH:menadione oxidoreductase were very resistant to such respiratory chain inhibitors as rotenone, capsaicin, and $AgNO_3$, whereas these activities were sensitive to 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Based on these results, we suggest that the aerobic respiratory chain-linked NADH oxidase system of B. cereus KCTC 3674 possesses an HQNO-sensitive NADH:quinone oxidoreductase that lacks an energy coupling site containing FAD as a cofactor.

Effect of Ginseng Saponins on $K^+-Dependent$ Phosphatase Activity of Dog Cardiac Sarcolemma (인삼 사포닌이 개 심실 형질막의 $K^+$-의존성 포스파타제 활성에 미치는 영향)

  • Lee, Shin-Woong;Lee, Jeung-Soo
    • YAKHAK HOEJI
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    • v.36 no.2
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    • pp.129-136
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    • 1992
  • The effects of ginseng saponins, gypsophila saponin, sodium dodecyl sulfate(SDS), and Triton X-100 on membrane $K^+-dependent$ phosphatase activity which is lipid dependent and represents dephosphorylation step of the complete Na+, $K^+-ATPase$ reaction were investigated in this study to elucidate whether the effects of ginseng saponins are due to the detergent action, using sarcolemma enriched preparation isolated from dog ventricle. $Na^+$, $K^+-ATPase$ and $K^+-dependent$ phosphatase activities of cardiac sarcolemma were about $143\;{\mu}mol$ Pi/mg protein/hr and $34\;{\mu}mol$ p-nitrophenol/mg protein/hr, respectively. While ginseng saponins (triol>total>diol) inhibited $K^+-dependent$ phosphatase activity, gypsophila saponin, and low dose of SDS($0.4\;{\mu}g/{\mu}g$ protein), and Triton X-100 ($0.6\;{\mu}g/{\mu}g$ protein) increased the enzyme activity, indicating disruptive effect of detergents on membrane barriers. The activating effect of low doses of Triton X-100 on membrane $K^+-dependent$ phosphatase appeared at concentration decreasing light scattering. However, the inhibitory effect of ginseng saponin appeared before a decrease in light scattering. These results suggest that low concentrations of ginseng saponins inhibit the membrane $K^+-dependent$ phosphatase by interacting directly with enzyme before membrane disruption.

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Purification of Band 3 from the Human Erythrocyte Membrane and its Incorporation into Liposome (사람 적혈구막 Band 3의 정제 및 Liposome으로의 도입)

  • Kim, Jae-Ryong;Kim, Jung-Hye;Lee, Ki-Yung
    • Journal of Yeungnam Medical Science
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    • v.3 no.1
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    • pp.41-48
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    • 1986
  • Band 3, the predominent 95,000 dalton anion transport protein, is the major intrinsic glycoprotein of the human erythrocyte membrane. This anion carrier exists as a dimer and binds the cytoskeletons such as spectrin, ankyrin and actin. And the liposomes are vesicular structures which form spontaneouly upon hydration of phospholipids. These artificial lipid vesicles have been investigated as model of the biological membranes and as a mean of improving the delivery of nucleic acids, drugs, proteins and biological substances to specific target tissues and cells. In this study, we were purified Band 3 from the human erythrocyte membrane(ghost) was prepared by hemolysis of intact human erythrocyte with weak alkali-hypotonic solution. Band 6 was removed from ghost by extracting with solution of an ionic strength of 0.15. Band 3 and Band 4 were solubilized selectively by extracting Band 6-depleted ghosts with Triton X-100 under nondenaturing conditions. Band 3 was then purified from Triton X-100 extract treated with p-chloromercuribenzoate by sucrose density gradient ultracentrifugation. This purified Band 3 was incorporated into liposomes prepared by reverse-phase evaporation. Phosphatidyl L-serine and cholesterol(1 : 1 molar ratio) were dissolved in chloroform and then chloroform was removed by rotatory evaporation under reduced pressure. Band 3 solution without Triton X-100 was introduced into a mixture of lipids and diethylether. Diethylether was subsequently removed by evaporation. This purified Band 3 and its incorporation into liposomes were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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