• BMB Reports
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• v.28 no.1
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• pp.40-45
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• 1995

Acetolactate synthase purified from Serratia marcescens ATCC 25419 was rapidly inactivated by the thiol specific reagent p-chloromercuribenzoate (PCMB), the tryptophan specific reagent N-bromosuccinimide (NBS), and the arginine modifying reagent phenylglyoxal (PGO). Inactivation by PCMB was prevented by both ${\alpha}$-ketobutyrate and pyruvate, and the second order rate constant for the inactivation was $2480\;M^{-1}{\cdot}min^{-1}$. The reaction order with respect to PCMB was 0.94. The inactivation of the enzyme by NBS was also substantially reduced by both ${\alpha}$-ketobutyrate and pyruvate. The second order rate constant for inactivation by NBS was $15,000\;M^{-1}{\cdot}min^{-1}$, and the reaction order was 2.0. On the other hand, inactivation by PGO was partially prevented by ${\alpha}$-ketobutyrate, but not by pyruvate. The second order rate constant for the inactivation was $1480\;M^{-1}{\cdot}min^{-1}$ and the order of reaction with respect to PGO was 0.75. These results suggest that essential cysteine, tryptophan and arginine are located at or near the substrate binding site.

• Clinical and Experimental Pediatrics
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• v.52 no.5
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• pp.567-575
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• 2009

Purpose : Epilepsy affects more than 0.5% of the world's population. It has a large genetic component and is caused by electrical hyperexcitability in the central nervous system. Despite its prevalence, the disease lacks definitive diagnostic serological biomarkers. To identify potential biomarkers for epilepsy by a convenient method, we analyzed the expression of serum proteins, reflecting alterations in the patient's proteomes. Methods : We compared two-dimensional electrophoretic band patterns of human sera from eight patients with temporal lobe epilepsy (TLE) with those of eight control subjects. The differentially expressed bands were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry. esults : Twelve proteins were differentially expressed in the TLE group, of which 6 were identified. Expression of haptoglobin Hp2, PRO2675, immunoglobulin heavy chain constant region gamma 2, an unnamed protein, and three unidentified proteins were upregulated in serum from the patients with TLE, whereas those of major histocompatibility complex (MHC) class I antigen, plasma retinol-binding protein precursor, and three unidentified proteins were downregulated in these patients. After resection of the epileptogenic zone, the expressions of MHC class I antigen, immunoglobulin heavy chain constant region gamma 2, two of the downregulated unidentified proteins, and one of the upregulated unidentified proteins returned to the normal range. Conclusion : The 12 serum proteins in this study are potentially useful biomarkers for the diagnosis and monitoring of TLE.

• BMB Reports
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• v.32 no.3
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• pp.294-298
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• 1999

Myo-inositol monophosphate phosphatase is a key enzyme in the phosphoinositide cell-signaling system. Incubation of myo-inositol monophosphate phosphatase from porcine brain with N-bromosuccinimide (NBS) resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order kinetics with the second-order rate constant of $3.8{\times}10^3\;M^{-1}min^{-1}$. The time course of the reaction was significantly affected by the substrate myo-inositol-1-phosphate, which afforded complete protection against the loss of catalytic activity. Spectrophotometric studies indicated that about one oxindole group per molecule of enzyme was formed following complete loss of enzymatic activity. It is suggested that the catalytic function of myo-inositol monophosphate phosphatase is modulated by the binding of NBS to a specific tryptophan residue at or near the substrate binding site of the enzyme.

• Biomolecules & Therapeutics
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• v.24 no.2
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• pp.191-198
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• 2016

The vitamin D receptor (VDR) is a member of the nuclear receptor (NR) superfamily. The VDR binds to active vitamin $D_3$ metabolites, which stimulates downstream transduction signaling involved in various physiological activities such as calcium homeostasis, bone mineralization, and cell differentiation. Quercetin is a widely distributed flavonoid in nature that is known to enhance transactivation of VDR target genes. However, the detailed molecular mechanism underlying VDR activation by quercetin is not well understood. We first demonstrated the interaction between quercetin and the VDR at the molecular level by using fluorescence quenching and saturation transfer difference (STD) NMR experiments. The dissociation constant ($K_d$) of quercetin and the VDR was $21.15{\pm}4.31{\mu}M$, and the mapping of quercetin subsites for VDR binding was performed using STD-NMR. The binding mode of quercetin was investigated by a docking study combined with molecular dynamics (MD) simulation. Quercetin might serve as a scaffold for the development of VDR modulators with selective biological activities.

• Animal cells and systems
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• v.1 no.4
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• pp.583-587
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• 1997

Incubation of an NADPH-dependent succinic semialdehyde reductase from bovine brain with N-bromosuccinimide (NBS) resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order kinetics with the second-order rate constant of $6.8\times{10}^3$ $M^-1$ $min^{-1}$. The inactivation was prevented by preincubation of the enzyme with substrate succinic semialdehyde, but not with coenzyme NADPH. There was a linear relation-ship between oxindole formation and the loss of enzyme activity. Spectro-photometric studies indicated that about one oxindole group per molecule of the enzyme was formed following complete loss of enzymatic activity. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of NBS to a specific tryptophan residue at or near the substrate binding site of the enzyme.

• Bulletin of the Korean Chemical Society
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• v.31 no.10
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• pp.2913-2917
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• 2010

When rubber dissolved in toluene was used as a binding material of graphite powder, the mechanical robustness of the carbon paste was guaranteed by the fast volatility of the solvent immediately after electrode construction. This characteristic of the rubber solution met qualifications for practical use of carbon paste electrodes and enabled the design of a new enzyme electrode bound with EPDM. In order to confirm whether the electrode shows quantitative electrochemical behaviors or not, its kinetic parameters, e. g. the symmetry factor (0.2), the exchange current density ($3.66\;{\mu}A/cm^2$), the capacity of the double layer ($2.0{\times}10^{-5}\;F$), the Michaelis constant ($4.39{\times}10^{-3}\;M$), the diffusion coefficient of substrate ($2.58{\times}10^{-12}\;cm^2/sec$), the time constant (0.018 sec) and other factors were investigated.

• Bulletin of the Korean Chemical Society
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• v.15 no.7
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• pp.549-553
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• 1994

The 2-(dimethylaminomethyl)thiophene (dmamt) complexes with copper(II) chloride and bromide were prepared and characterized by optical, EPR, XPS spectroscopies and magnetic susceptibility measurements. The low-energy absorption band above 850 nm and the relatively small EPR hyperfine coupling constant ($A_{//}{\simeq}$125 G) indicate the pseudotetrahedral site symmetry around copper(II) ion both in Cu(dmamt)$Cl_2$ and Cu(dmamt)$Br_2$ complexes. The higher satellite to main peak intensity of Cu $2P_{3/2}$ core electron binding energy in XPS spectra also supports the pseudotetrahedral geometry around the copper(II) ions having $CuNSX_2$ chromophores. The distortion from square-planar to pseudotetrahedral symmetry is likely to arise from the steric hindrance of the bulky dmamt ligand in the complex. Magnetic susceptibility study shows that these compounds follow Curie-Weiss law in the temperature range of 77-300 K with positive Weiss constant exhibiting the ferromagnetic interaction between copper(II) ions in solid state.

• Journal of Life Science
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• v.30 no.11
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• pp.1012-1020
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• 2020

Remicade is a therapeutic biosimilar natural antibody in which the mouse variable domain has been linked to the human constant domain. It is a chimeric monoclonal antibody specific to tumor necrosis factor-alpha (TNF-α) and has been developed for the treatment of rheumatoid arthritis. To investigate the biological activity of the Remicade antibody, we carried out a bioinformatics study using a protein data bank to characterize the TNF-α antigen binding mechanism of the Remicade natural antibody. Because the production of the Remicade antibody is often limited by genetic instability of the natural antibody-producing cell, we generated a Remicade single-chain variable domain fragment antibody (Remicade) in which a heavy chain variable domain (VH) is joined with a light chain variable domain (VL) by a polypeptide linker. Furthermore, Remicade was fused to a leucine zipper (RemicadeScZip) for higher production and higher antigen-binding activity than Remicade. The Remicade and Remicade ScZip were expressed in Escherichia coli and purified by a Ni⁺-NTA-agarose column. As expected, the purified proteins had migrated as 28.80 kDa and 33.96 kDa in sodium dodecyl sulfate-polyacrylamide electrophoresis. The TNF-α antigen binding activity of Remicade was not observed by ELISA and western blot. In contrast, RemicadeScZip showed antigen-binding activity. Additional bio-layer interferometry analysis confirmed the antigen-binding activity of RemicadeScZip, suggesting that the leucine zipper stabilized the folding of RemicadeScZip in a denatured condition and improved the TNF-α antigenbinding activity.

• Biomolecules & Therapeutics
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• v.3 no.4
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• pp.266-272
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• 1995

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently through the use of specific antibodies. To generate and characterize a moloclonal antibody against $\beta$-adrenergic receptor, a synthetic $\beta$2-adrenergic receptor peptide (Phe-Gly-Asn-Phe-Trp-Cys-Phe-Trp-Thr-Ser-lle-Asp-Val-Leu) which may comprise part of $\beta$-adrenergic receptor ligand binding pocket was coupled to Keyhole Limpet Hemocyanin (KLH) and used as an immunogen. Male BALB/C mice were immunized with this antigen and the immunized spleen was fused with myeloma SP2/0-Ag14 cells to produce monoclonal antibodies. Two clones were obtained but one of monoclonal antibodies, mAb5G09, was used throughout in this study because the other clone, mAb5All showed weak immunoreactivity against KLH as well. The mouse monoclonal antibody mAb5G09 produced in this study showed immunoreactivity to peptide-KLH conjugates and also to human A43l cells and guinea pig lung $\beta$2-adrenergic receptor as revealed by ELISA and western blot. In the course of determination of the effects of mAb5G09 on $\beta$-receptor ligand binding, it was observed that mAb5G09 specifically bound $\beta$-adrenergic radioligand [$^3$H]dihydroalprenolol (DHA) with a dissociation constant (Kd) of 60 nM. The [$^3$H]DHA binding activity of mAb5G09 had characteristics of immunoglobulins and the binding activity was not observed in the control anti-KLH monoclonal antibody. The monoclonal antibody, mAb5G09 produced in this study may provide useful models for the study of the structure of receptor binding sites.

• Bulletin of the Korean Chemical Society
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• v.24 no.5
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• pp.547-551
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• 2003

In the present work, the interaction of three water soluble porphyrins, tetra(p-trimethyle) ammonium phenyl porphyrin iodide (TAPP) as a cationic porphyrin, tetra sodium meso-tetrakis (p-sulphonato phenyle) porphyrin (TSPP) as an anionic porphyrin and manganese tetrakis (p-sulphonato phenyl) porphinato acetate (MnTSPP) as a metal porphyrin, with histone H₂B have been studied by isothermal titration microcalorimetry at 8 mM phosphate buffer, pH 6.8 and 27 °C. The values of binding constant, entropy, enthalpy and Gibbs free energy changes for binding of the first MnTSPP, and first and second TSPP and TAPP molecules were estimated from microcalorimetric data analysis. The results represent that the process is both entropy and enthalpy driven and histone induces self-aggregation of the porphyrins. The results indicate that both columbic and hydrophobic interactions act as self-aggregation driving forces for the formation of aggregates around histone.