• BMB Reports
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• v.30 no.5
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• pp.308-314
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• 1997

We investigated biotinylation of nitrocellulose membrane for immuno-filtration capture assay. In order to enhance the efficiency of biotinylation, nitrocellulose membranes were pretreated with several chemicals for the purpose of suitable protein absorption through surface modification. As a signal generating enzyme, urease was used and the concentration of avidin was optimized for the efficient binding kinetics between urease-biotin in liquid phase and biotinylated membrane in solid phase. For effective biotinylation, bovine serum albumin-biotin complexes could be immobilized at a concentration of $370\;{\mu}g$/stick ($4.4\;cm^2$). Among tested chemicals, polylysine (0.25%) showed a significant effect in biotinylation. Polylysine is thought to enhance surface area by extending unbound residues into solution. Time of treatment over 30 min and higher molecular weight of polylysines (58,100 dalton) showed positive effect on the enhancement of biotinylation. The result from this study may be useful for developing a new biosensor and other biofunctional membranes for examining molecular recognition.

• Environmental Engineering Research
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• v.17 no.3
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• pp.145-150
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• 2012

Pb-contaminated soil at a clay shooting range was analyzed by the sequential extraction method to identify metal binding properties in terms of detrital and non-detrital forms of the soil. Most of the metals in the soils existed as non-detrital forms, exchangeable and carbonate-bound forms, which could be easily released from the soil by a washing method. Therefore, the characteristics of Pb desorption for remediation of the Pb-contaminated soil were evaluated using hydrochloric acid (HCl) by a washing method. Batch experiments were performed to identify the factors influencing extraction efficiency. The effects of the solid to liquid (S/L) ratio (1:2, 1:3, and 1:4), soil particle size, and extraction time on the removal capacity of Pb by HCl were evaluated. Soil samples were collected from two different areas: a slope area (SA) and a land area (LA) at the field. As results, the optimal conditions at 2.8 to 0.075 mm of particle size were 1:3 of the S/L ratio and 10 min of extraction time for SA, and 1:4 of the S/L ratio and 5 min of extraction time for LA. The characteristics of Pb desorption were adequately described by two-reaction kinetic models.

• Microbiology and Biotechnology Letters
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• v.22 no.6
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• pp.636-642
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• 1994

Essential amino acids involving in the catalytic mechanism of the $\beta$-D-xylosidase of Bacillus stearothermophilus were determined by chemical modification studies. Among various che- mical modifiers tested N-bromosuccinimide (NBS), $\rho$-hydroxymercurybenzoate (PHMB), N-ethylma- leimide, 1-[3-(di-ethylamino)-propyl]$-3-ethylcarbodi-imide (EDC), and Woodward's Reagent K(WRK)inactivated the enzyme, resulting in the residual activity of less than 20%. WRK reduced the enzyme activity by modifying carboxylic amino acids, and the inactivation reacion proceeded in the form of pseudo-first-order kinetics. The double-lagarithmic plot of the observed pseudo-first- order rate constant against the modifier concentration yielded a reaction order of 2, indicating that two carboxylic amino acids were essential for the enzyme activity. The $\beta$-D-xylosidase was also inactivated by N-ethylmaleimide which specifically modified a cysteine residue with a reaction order of 1, implying that one cysteine residue was important for the enzyme activity. Xylobiose protected the enzyme against inactivation by WRK and N-ethylmaleimide, revealing that carboxylic amino acids and a cysteine residue were present at the substrate-binding site of the enzyme molecule.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.75-75
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• 1995

Succinic semialdehyde reductase, one of key enzyme of GABA shunt in CNS, is inactivated by o-phthalaldehyde, The inactivation followed pseudo first-order kinetics, and the second-order rate constant for the inactivation process was 28 M$\^$-1/s$\^$-1/ at pH 7.4 and 25$^{\circ}C$. The absorption spectrum(λ$\_$max/=377nm), fluorescence exitation(λ$\_$max/=340nm) and fluorescence emission spectra (λ$\_$max/=409nm) were consistent with the formation of an isoindole derivative in the catalytic site between a cysteine and a lysine residues about 3${\AA}$ apart. The substrate, succinic semialdehyde, did not protect the enzymatic activity against inactivation, whereas the coenzyme, NADPH, protected against o-phthalaldehyde induced inactivation of the enzyme. About 1 isoindole group per moi of the enzyme was formed following complete loss of the enzymatic activity. These results suggest that the amino acid residues of the enzyme participating in reaction with o-phthalaldehyde more likely residues at or near the coenzyme binding site.

• Bulletin of the Korean Chemical Society
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• v.35 no.2
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• pp.425-430
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• 2014

Amyloidogenic proteins often exhibit fibrillar polymorphism through alternative assembly processes, which has been considered to have possible pathological implications. Here, firefly luciferase (LUC) is shown to induce amyloid polymorphism of ${\alpha}$-synuclein, the major constituent of Lewy bodies found in Parkinson's disease, by acting as a novel template. The drastically accelerated fibrillation kinetics of ${\alpha}$-synuclein with LUC required the nucleation center produced by the active enzyme of LUC. Fluorescent dye binding, transmission electron microscopy, and Fourier transformed infrared spectroscopy revealed the morphologically distinctive amyloid fibrils of ${\alpha}$-synuclein prepared in the absence or presence of LUC. As the altered morphological characteristics became inherent to the mature fibrils, those properties were inherited to next-generations via nucleation-dependent fibrillation process. The seed control, therefore, would be an effective means to modify amyloid fibrils with different biochemical characteristics. In addition, the LUC-directed amyloid fibrillar polymorphism also suggests that other cellular biomolecules including enzymes in general are able to diversify amyloid fibrils, which could be self-propagated with diversified biological activities, if any, inside cells.

• Journal of Environmental Health Sciences
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• v.19 no.4
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• pp.83-89
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• 1993

To evaluate an effect of methanethiol on a cause of erythrocyte membrane damage in rats, methanethiol was given at 11.25 rag/100 g body weight, and after 4 hr, the animals were sacrifled, the activities of Na$^+$/K$^+$ ATPase, protein contents in partial purified erythrocyte membrane and erythrocyte indices were determined Concomitantly, in vitro, effect of methanethiol on the erythrocyte fragility, Na$^+$/K$^+$ ATPase activity and its kinetics in various concentration of substrate from the preincubated erythrocyte membrane with methanethiol were demonstrated. The spleen weight per body weight (%) and MCV of erythrocyte in methanethiol-treated rats were more increased than those in the control group. The Na$^+$/K$^+$ ATPase activities in erythrocyte membrane were more decreased in methanethiol-treated rats than those in the control group. The apply of 0.05 ng rat whole blood to the 0.24 mg/ng of methanethiol solution in isotonic condition showed the complete hemolysis. The Na$^+$/K$^+$ ATPase activity in preincubated erythrocyte membrane with methanethiol at 37$\circ$C showed the dual effect and the K$_m$ value of Na$^+$/K$^+$ ATPase was higher in the preincubated erythrocyte membrane with methanethiol than that in the preincubated erythrocyte membrane omitted the methanethiol. These results suggest that the methanethiol may induce the damage of rat's erythrocyte membrane due to a change in substrate binding affinity of Na$^+$/K$^+$ ATPase.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.268-273
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• 2019

The specificity of a Bacillus licheniformis uridine diphosphate (UDP) glycosyltransferase, YjiC, was increased towards thymidine diphosphate (TDP)-sugar by site-directed mutagenesis. The Arg-282 of YjiC was identified and investigated by substituting with Trp. Conversion rate and kinetic parameters were compared between YjiC and its variants with several acceptor substrates such as 7-hydroxyflavone (7-HF), 4',7-dihydroxyisoflavone, 7,8-dihydroxyflavone and curcumin. Molecular docking of TDP-glucose and 7-HF with YjiC model showed pi-alkyl interaction with Arg-282 and His-14, and pi-pi interaction with $His^{14}$ and thymine ring. YjiC (H14A) variant lost its glucosylation activity with TDP-glucose validating significance of His-14 in binding of TDP-sugars.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.84-90
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• 1994

Kinetic parameters of various substrates and inhibitors were measured to elucidate the binding requirements of the active site of intracellular adenosine deaminase (ADA) in Aspergillus oryzae. 3'-Deoxyadenosine was the best substrate according to the value of relative kcat/$K_m$. Purine riboside was found to be the strongest inhibitor with the $K_i$ value of $3.7{\times}10^{-5}$M. Adenine acted neither as a substrate nor as an inhibitor, suggesting the presence of ribose at N-9 of adenosine was crucial to binding. ADA also catalyzed the dechlorination of 6-chloropurine riboside, generating inosine and chloride ions. Substrate specificity of 6-chloropurine riboside was 0.86% of adenosine. Purine riboside, a competitive inhibitor of ADA, inhibit the dechlorination with similar $K_i$ value, suggesting that the same binding site was involved in deamination and dechlorination. Among the sulfhydryl group reagents, mercurials, pchloromercuribenzoate (PCMB), mersalyl acid and $HgCl_2$ inactivated the enzyme. Mersalyl acid-inactivated ADA was reactivated by thiol reagents, but PCMB-inactivated enzyme was not. When ADA was treated with the mercurial reagents, the inhibition constants and inhibition patterns were determined. Each inhibition was competitive with substrate. The $K_i$ values of these mercurial reagents were lower in 10 mM phosphate buffer than in 100 mM phosphate buffer, showing phosphate dependency.

• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1138-1149
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• 2017

The use of microalgal biomass is an interesting technology for the removal of heavy metals from aqueous solutions owing to its high metal-binding capacity, but the interactions with bacteria as a strategy for the removal of toxic metals have been poorly studied. The goal of the current research was to investigate the potential of Burkholderia tropica co-immobilized with Chlorella sp. in polyurethane discs for the biosorption of Hg(II) from aqueous solutions and to evaluate the influence of different Hg(II) concentrations (0.041, 1.0, and 10 mg/l) and their exposure to different contact times corresponding to intervals of 1, 2, 4, 8, 16, and 32 h. As expected, microalgal bacterial biomass adhered and grew to form a biofilm on the support. The biosorption data followed pseudo-second-order kinetics, and the adsorption equilibrium was well described by either Langmuir or Freundlich adsorption isotherm, reaching equilibrium from 1 h. In both bacterial and microalgal immobilization systems in the co-immobilization of Chlorella sp. and B. tropica to different concentrations of Hg(II), the kinetics of biosorption of Hg(II) was significantly higher before 60 min of contact time. The highest percentage of biosorption of Hg(II) achieved in the co-immobilization system was 95% at pH 6.4, at 3.6 g of biosorbent, $30{\pm}1^{\circ}C$, and a mercury concentration of 1 mg/l before 60 min of contact time. This study showed that co-immobilization with B. tropica has synergistic effects on biosorption of Hg(II) ions and merits consideration in the design of future strategies for the removal of toxic metals.

• BMB Reports
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• v.40 no.1
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• pp.100-106
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• 2007

The wound-inducible lipoxygenase obtained from maize is one of the nontraditional lipoxygenases that possess dual positional specificity. In this paper, we provide our results on the determination and comparison of the kinetic constants of the maize lipoxygenase, with or without detergents in the steady state, and characterization of the dependence of the kinetic lag phase or initial burst, on pH, substrate, and detergent in the pre-steady state of the lipoxygenase reaction. The oxidation of linoleic acid showed a typical lag phase in the pre-steady state of the lipoxygenase reaction at pH 7.5 in the presence of 0.25% Tween-20 detergent. The reciprocal correlation between the induction period and the enzyme level indicated that this lag phenomenon was attributable to the slow oxidative activation of Fe (II) to Fe (III) at the active site of the enzyme as observed in other lipoxygenase reactions. Contrary to the lagging phenomenon observed at pH 7.5 in the presence of Tween-20, a unique initial burst was observed at pH 6.2 in the absence of detergents. To our knowledge, the initial burst in the oxidation of linoleic acid at pH 6.2 is the first observation in the lipoxygenase reaction. Kinetic constants (Km and kcat values) were largely dependent on the presence of detergent. An inverse correlation of the initial burst period with enzyme levels and interpretations on kinetic constants suggested that the observed initial burst in the oxidation of linoleic acid could be due to the availability of free fatty acids as substrates for binding with the lipoxygenase enzyme.