• Analytical Science and Technology
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• v.13 no.5
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• pp.624-629
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• 2000

A simple and rapid homogeneous enzyme-linked binding assay for mistletoe lectin I(ML I) was developed using a coupled enzyme system of malate dehydrogenase (MDH) and D-galactose. A highly substituted MDH-galactose conjugate was prepared by employing an isothiocyanate method for formation of thiourea bond. In the presence of ML I, ML I inhibits the activity of the conjugate based on the ML I/D-galactose specific interaction. Thus, the concentration of ML I can be related to the homogeneous inhibition of the MDH-galactose conjugate. Using this method. ML I can be measured at the level of microgram per milliliter within 10 minutes.

• Journal of Korean Society on Water Environment
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• v.25 no.1
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• pp.84-89
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• 2009

Our nation's water resources remain susceptible to contamination by phenolic agrichemicals. These compounds can be toxic to a variety of organisms including humans. Their disposal is restricted in many countries with strict limits for acceptable concentrations in drinking water. Enzyme-mediated in situ stabilization has been advocated as an approach for the treatment of phenolic compounds in soils and groundwater. This study reports the development of a new approach to quantify the activity of the HRP enzyme in aqueous systems. The method is based on the coupled processes of energy transfer and enhanced chemiluminescence using a luminol-$H_2O_2$-HRP system. In this study, the effects of solution pH, ionic strength and aqueous concentrations of HRP, $H_2O_2$ and enhancer were evaluated on the p-iodophenol-enhanced, HRP-catalyzed chemiluminescence reaction intensity in Tris-HCl buffer. All assay components were found to affect the maximum chemiluminescene intensity. The calibration curve for HRP showed the linear relationship with maximum light intensity.

• Analytical Science and Technology
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• v.8 no.3
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• pp.359-364
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• 1995

Urinary dipeptidase(Udpase) was assayed by fluorometric analysis of NADH which was produced from an indicator enzyme, L-alanine dehydrogenase. The reaction mixture was consisted of a dipeptide(L-ala-L-ala), ${\beta}-NAD^+$, L-alanine dehydrogenase in 12.5 mM sodium carbonate buffer, pH 9.0, and urinary dipeptidase which initiated the reaction. The fluorescence intensity of NADH was measured as a function of time with the excitation wavelength at 340nm and emission at 460nm. Comparison of this fluorometric method with the conventional spectrophotometric method utilizing glycyldehydrophenylalanine(Gdp) as substrate provided the correlation coefficient of 0.996 and increased the sensitivity more than ten times.

• Journal of Microbiology and Biotechnology
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• v.33 no.2
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• pp.228-234
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• 2023

In this study, the performance of a gold biosensor combined with light microscope imaging system (GB-LMIS) was comparatively evaluated against enzyme-linked immunosorbent assay (ELISA) for detecting Salmonella under simulated chilling condition. The optimum concentration of antiSalmonella polyclonal antibodies (pAbs) was determined to be 12.5 and 100 ㎍/ml for ELISA and GBLMIS, respectively. GB-LMIS exhibited a sufficient and competitive specificity toward three tested Salmonella among only. To mimic a real-world situation, chicken was inoculated with Salmonella cocktail and stored under chilling condition for 48 h. The overall growth of Salmonella under chilling condition was significantly lower than that under non-exposure to the chilling condition (p < 0.05). No significant differences in bacterial growth were observed between brain heart infusion and brilliant green broth during the enrichment period (p > 0.05). Finally, both GB-LMIS and ELISA were employed to detect Salmonella at every 2-h interval. GB-LMIS detected Salmonella with a competitive specificity by the direct observation of bacteria on the sensor using a charge-coupled device camera within a detection time of ~2.5 h. GB-LMIS is a feasible, novel, and rapid method for detecting Salmonella in poultry facilities.

• Proceedings of the Korean Biophysical Society Conference
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• 1999.06a
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• pp.49-49
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• 1999

$H^{＋}$-ATPases play major roles in various cellular physiology. In order to characterize the effects of heavy metal ions on the activity of $H^{＋}$-ATPases, microsomes were isolated from the roots of tomato grown hydroponically. The activity of microsomal $H^{＋}$-ATPase was measured by an enzyme-coupled assay. H $g^{2＋}$ inhibited the activity of microsomal $H^{+}$-ATPase as a dose-dependent manner, F $e^{3+}$ and Z $n^{2+}$ inhibited the activity although they also blocked the activities of enzymes used in the assay, and C $s^{+}$ and $Ba^{2+}$ showed no significant effect.(omitted)d)ted)d)

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.4
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• pp.344-350
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• 2006

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at $59^{\circ}C$ and pH 2. Along with another thermoacidophilic archaeon, Sulfolobus solfataricus, it is known to metabolize glucose by the non-phosphorylated Entner-Doudoroff (nED) pathway. In the course of these studies, the specific activities of glyceraldehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher in T. acidophilum than in S. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity from T. acidophilum cell extracts. The N-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49kDa and followed Michaelis-Menten kinetics with $K_m$ values of 0.56 and 0.32mM for DL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at $70^{\circ}C$. Of the seven sugars and four phosphate donors tested, only DL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as $Mn^{2+},\;CO^{2+},\;Ni^{2+},\;Zn^{2+},\;Ca^{2+},\;and\;Sr^{2+}$, were substituted for $Mg^{2+}$ the enzyme activities were less than 10% of that obtained in the presence of $Mg^{2+}$. The amino acid sequence of T. acidophilum glycerate kinase showed no similarity with E. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.

• Biomedical Science Letters
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• v.6 no.4
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• pp.261-268
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• 2000

Fibrinolytic enzyme (FE-2) was purified from the fruiting bodies of Tricholoma saponaceum using DEAE-Cellulose chromatography and Mono-S column chromatography, The enzyme has a molecular weight of 18.23 kDa and include Zn$^{2+}$ ion as found by ICP/MS. The N-terminal amino acid sequence of the enzyme was A-L-Y-V-G-X-S-P-X-Q-Q-S-L-L-V It has a pH optimum at pH 7.5, suggested that FE-2 was a neutral pretense. The activity of FE-2 was highly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. The activity of FE-2 was increased by $Mg^{2+}$, Zn$^{2+}$, Fe$^{2+}$, and Co$^{2+}$, but the enzyme activity was totally inhibited by Hg$^{2+}$. No inhibition was found with PMSF, E-64, pepstatin and 2-mercaptoethanol. The enzyme hydrolyzed both $A\alpha$ and B$\beta$ chains of human fibrinogen. The $\gamma$ chain was resistant to hydrolysis by FE-2.

• Applied Biological Chemistry
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• v.42 no.4
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• pp.298-303
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• 1999

In order to characterize the effects of heavy metal ions on the microsomal ATPase activities, microsomes were prepared from the roots of tomato plant and the activity of microsomal ATPase was measured by an enzyme-coupled assay. $Hg^{2+}$ inhibited the activity of microsomal ATPase in a dose-dependent manner, while $Gd^{3+}$, $Fe^{3+}$, $La^{3+}$, $Zn^{2+}$, and $Pb^{2+}$ inhibited not only the ATPase activity but also the activities of enzymes used in the assay. However, $Cs^+$ and $Ba^{2+}$ showed no significant effect. $Hg^{2+}$ inhibited the activities of both plasma membrane and vacuolar membrane $H^+-ATPases$. In the dose-response to $Hg^{2+}$, the activities of both microsomal $H^+-ATPases$ were severely inhibited at the concentration of $Hg^{2+}$ above $10\;{\mu}M$ and were completely inhibited at 1 mM $Hg^{2+}$. Apparent Ki values of $Hg^{2+}$ on the inhibitions of plasma membrane and vacuolar membrane $H^+-ATPases$ were $80\;{\mu}M$ and $58\;{\mu}M$, respectively. The $Hg^{2+}$-induced inhibitions were reversible since the addition of dithiothreitol completely reversed the inhibitory effects of $Hg^{2+}$. These results suggest that the inhibitory effects of $Hg^{2+}$ on both plasma, membrane and vacuolar membrane $H^+-ATPases$ are nonselective and reversible.

• Molecules and Cells
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• v.45 no.7
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• pp.495-501
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• 2022

Leucine dehydrogenase (LDH, EC 1.4.1.9) catalyzes the reversible deamination of branched-chain L-amino acids to their corresponding keto acids using NAD⁺ as a cofactor. LDH generally adopts an octameric structure with D4 symmetry, generating a molecular mass of approximately 400 kDa. Here, the crystal structure of the LDH from Pseudomonas aeruginosa (Pa-LDH) was determined at 2.5 Å resolution. Interestingly, the crystal structure shows that the enzyme exists as a dimer with C2 symmetry in a crystal lattice. The dimeric structure was also observed in solution using multiangle light scattering coupled with size-exclusion chromatography. The enzyme assay revealed that the specific activity was maximal at 60℃ and pH 8.5. The kinetic parameters for three different amino acid and the cofactor (NAD⁺) were determined. The crystal structure represents that the subunit has more compact structure than homologs' structure. In addition, the crystal structure along with sequence alignments indicates a set of non-conserved arginine residues which are important in stability. Subsequent mutation analysis for those residues revealed that the enzyme activity reduced to one third of the wild type. These results provide structural and biochemical insights for its future studies on its application for industrial purposes.

• Journal of Acupuncture Research
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• v.17 no.3
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• pp.176-187
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• 2000

Objectives : This study was purposed to investigate the antioxidative effects of Paeoniae radix aqua-acupuncture solution(PR) on culture liver cell system, lipid peroxidation and antioxidative enzyme activities in tert-butyl hydroperoxide(t-BHP) treatmented conditions. Methods : Cultured normal rat liver cell(Ac2F) were prepared and incubated with or without PR(at 2% volume in culture medium). After 16~18hr, cells placed in DMEM medium without serum, and then incubated with 1mM t-BHP for 2hr. Viable cells were detected by MTT assay, and the levels of lipid peroxide(LPO) were measured by TBA method. And catalase activity was measured as the decrease in hydrogen peroxide absorbance at 240nm on spectrophotometer using 30mM hydrogen peroxide. Superoxide dismutase(SOD) were assayed by recording the inhibition of nitro blue tetrazolium reduction with xanthine and xanthine oxidase. Glutathione peroxidase(GPX) activity was determined by the modified coupled assay developed by Paglia and Lawrence. The reaction was started by addition of 2.2mM hydrogen peroxide as substrate. The change in absorbance at 340nm was measured for 1min on spectrophotometer. Glutathione-S-transferase(GST) activity was assayed with CDNB as substrate and enzyme activity of GST towards the glutathione conjugation of CDNB. Results : Cell killing was significantly enhanced by addition of t-BHP compared to those of untreated group. PR pretreated cell resisted the toxic effects of t-BHP. LPO levels of t-BHP treatment group were significantly higher than other groups. This increased level was significandy reduced by PR pretreatment. The t-BHP treatment resulted in a decrease of catalase, GPX and GST activities. By contrast, PR pretreatment markedly increased compare to those of untreated groups. Conclusions : T-BHP which can produce intracellular free radical was used for inducer of the peroxidation of cellular lipids. PR protected the cell death induced by t-BHP and significantly increased cell viabiliry in the normal rat liver cell, and showed effective inhibition of lipid peroxidation, and elevations of catalase, GPX and GST activities. These results suggested that PR might play a protective role in lipid peroxidation by free radicals.