• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1320-1324
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• 2006

The pancreatic and neutrophil elastases are associated with several illnesses including lung and vascular diseases, various cancers, and pancreatitis. The development of a potent and specific inhibitor to the elastases could lead to new therapies. In this study, an agar diffusion method was modified to include a substrate-dye conjugate (Elastin-Congo red) as a substrate of elastase and an indicator of elastase inhibitory activity. The Elastin-Congo red agar plates consisted of 0.1 % Elastin-Congo red and 2.5% agar. The elastase and elastase inhibitors were simultaneously loaded into wells, ultimately resulting in halo formations in which the halo diameter decreased as the concentration of elastase inhibitor increased. The concentration of elastase inhibitor in the samples, therefore, was inversely proportional to the halo diameters. This simplified method provided an excellent correlation with the standard microplate technique, which uses a chromogenic substrate. The concentration of elastase inhibitor obtained from the culture supernatant of a recombinant elastase inhibitor produced by the yeast Pichia pastoris was easily determined. This study has established a simple modified and inexpensive agar diffusion method that is potentially useful for the identification, quantification, and screening of new elastase inhibitors.

• Bulletin of the Korean Chemical Society
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• 제20권3호
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• pp.301-306
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• 1999

Hepatitis C virus (HCV) has been known to be an enveloped virus with a positive strand RNA genome and the major agent of the vast majority of transfusion associated cases of hepatitis. For viral replication, HCV structural proteins are first processed by host cell signal peptidases and NS2/NS3 site of the nonstructural protein is cleaved by a zinc-dependent protease NS2 with N-terminal NS3. The four remaining junctions are cleaved by a separate NS3 protease. The solution conformations of NS4B/5A, NS5A/5B substrates and NS5A/5B inhibitor have been determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. NMR data suggested that the both NS5A/5B substrate and inhibitor appeared to have a folded tum-like conformation not only between P1 and P6 position but also C-terminal region, whereas the NS4B/5A substrate exhibited mostly extended conformation. In addition, we have found that the conformation of the NS5A/5B inhibitor slightly differs from that of NS5A/5B substrate peptide, suggesting different binding mode for NS3 protease. These findings will be of importance for designing efficient inhibitor to suppress HCV processing.

• Biomolecules & Therapeutics
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• 제13권1호
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• pp.32-34
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• 2005

Effect of isoliquiritigenin isolated from the heartwood of Dalbergia odorifera T. Chen (Leguminosae) on mushroom tyrosinase activity was investigated in vitro using L-tyrosine and L-3, 4-dihydroxyphenylalanine (L-DOPA) as the substrates. When L-tyrosine was used as a substrate, both isoliquiritigenin and kojic acid, a positive control, inhibited tyrosinase activity in a concentration-dependent manner. IC$_{50}$ values of isoliquiritigenin and kojic acid were 61.4 and 52.2 ${\muM}$, respectively. However, isoliquiritigenin showed week inhibitory effect on the oxidation of L-DOPA by tyrosinase with inhibition ratio of 9.1 ${\pm}$ 7.1% at 100 ${\muM}$. It is also suggested that 3-unsubstituted and 4-hydroxyl phenyl group in isoliquiritigenin plays an important role on the inhibition of tyrosinase activity when L-tyrosine was used as a substrate. Analysis of Lineweaver-Burk plot showed that isoliquiritigenin acts as a competitive inhibitor in case of L-tyrosine as a substrate.

• 한국동물학회지
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• 제39권2호
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• pp.190-197
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• 1996

The optimal conditions and substrate specificity of whole body esterase (EST) activity, effects of inhibitors (Eserine, Paraoxon, p-HMB, DDVP, DFP) on the enzyme, and ontogenv of the isozymes were determined in Lucilio ilfustris Meisen. The optimal temperature was $45^{\circ}C$ regardless of kind of reacted substrate, $\alpha-naphthyl$ acetate $(\alpha-Nal,$ a.naphthvl butylate $(\alpha-N),$ and Pnaphthyl acetate $(\beta-Na),$ but the optimal pH showed some regioselectivitv to naphthvl group of the esters; PH 7.0 for Iform, pH 7.5 for a-form. The maximum reaction rate was recorded at about 2.5 $\times$ 10's M of PNa and etNa, but 1.0 $\times$ 10'S M of $\alpha-Nb.$ Among the five EST inhibitors tested, DDVP was the most powerful. However, distinction of the relative specificity of inhibitors between three body parts, head, thorax, and abdomen, was shouts, representing differences in the distribution and activity of isozvmes. Of 12 carboxyl-esterases (CE), 8 cholinesterases (ChE) and 2 arvlesterases (ArE) identified based on their inhibitor specificity throughout the development, two larval and prepupal stage specific ChEs, no pupal specific, and 2 CEs.2ChEs. and one ArE adult specific isozvmes were confirmed.

• Bulletin of the Korean Chemical Society
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• 제32권4호
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• pp.1241-1248
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• 2011

Three dimensional quantitative structure-activity relationships (3D-QSARs) between new thiosemicarbazone analogues (1-31) as a substrate molecule and their inhibitory activity against tyrosinase as a receptor were performed and discussed quantitatively using CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) methods. According to the optimized CoMSIA 2 model obtained from the above procedure, inhibitory activities were mainly dependent upon H-bond acceptor favored field (36.5%) of substrate molecules. The optimized CoMSIA 2 model, with the sensitivity of the perturbation and the prediction, produced by a progressive scrambling analysis was not dependent on chance correlation. From molecular docking studies, it is supposed that the inhibitory activation of the substrate molecules against tyrosinase (PDB code: 1WX2) would not take place via uncompetitive inhibition forming a chelate between copper atoms in the active site of tyrosinase and thiosemicarbazone moieties of the substrate molecules, but via competitive inhibition based on H-bonding.

• BMB Reports
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• 제31권1호
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• pp.25-30
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• 1998

Kinetic studies of L-Alanine dehydrogenase from Bacillus subtilis-catalyzed reactions in the presence of $Zn^{2+}$ were carried out. The substrate (L-alanine) saturation curve is hyperbolic in the absence of the metal ion but it becomes sigmoidal when $Zn^{2+}$ is added to the reaction mixture indicating the positive cooperative binding of the substrate in the presence of zinc ion. The cooperativity of substrate binding depends on the xinc ion concentration: the Hill coefficients ($n_H$) varied from 1.0 to 1.95 when the zinc ion concentration varied from 0 to $60\;{\mu}m$. The inhibition of AlaDH by $Zn^{2+}$ is reversible and noncompetitive with respect to $NAD^+$ ($K_i\;=\;5.28{\times}10^{-5}\;M$). $Zn^{2+}$ itself binds to AlaDH with positive cooperativity and the cooperativity is independent of substrate concentration. The Hill coefficients of substrate biding in the presence of $Zn^{2+}$ are not affected by the enzyme concentration indicating that $Zn^{2+}$ binding does not change the polymerization-depolymerization equilibria of the enzyme. Among other metal ions, $Zn^{2+}$ appears to be a specific reversible inhibitor inducing conformational change through the intersubunit interaction. These results indicate that $Zn^{2+}$ is an allosteric competitive inhibitor and substrate being a non-cooperative per se, excludes the $Zn^{2+}$ from its binding site and thus exhibits positive cooperativity. The allosteric mechanism of AlaDh from Bacillus subtilis is consistent with both MWC and Koshland's allosteric model.

• 미생물학회지
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• 제31권2호
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• pp.148-156
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• 1993

Kinetic parameters of purine nucleoside phosphorylase (PNP) from Saccharomyces cerevisiae were measured. The Michaelis constants determined for substrates of the enzyme were $ 2.0 * 10^{-4}$ M for inosine, $2.0 *10^{-3}$ M for deoxyinosine, $ 2.0 * 10^{-5}$ M for guanosine and $2.0 10 ^{-5}$ M for deoxyguanosine. According to the ratio of relative $K_{cat}$Km, substrate specificity of each nucleoside was in the order of guanosine or deoxyguanosine, inosine and deoxyinosine. Cosubstrate, phosphate, revealed downward curvature in Lineweaver-Burk plot at high concentrations, indicating a negative cooperativity between subunits. The inhibition constants for purine analogs were measured to be $ 6 * 10^{-4}$ M for formycin B as the competitive inhibitor of inosine, $ 9 * 10^{-6}$ M for guanine as the competitive inhibitor of guanosine, $2 * 10^{-4}$ M for hypoxanthine as the non competitive inhibitor of guanosine and $4.5 * 10 ^{-4}$ M for 6-mercaptopurine as the non competitive inhibitor of guanosine. Alternative substrates, guanosine, deoxyguanosine and adenosine were found to act as competitive inhibitors with Ki values o $f^ 2.0 * 10 {-5}$ M, $2.6 * 10^{-5}$ M and $8.5 * 10 ^{-4}$ M, respectively, when inosine was the variable substrate. Guanosine and deoxyguanosine were also observed as competitive inhibitors with the Ki values of $1.8 * 10^{-5}$ M and $ 3.0 * 10^{-5}$ M, respectively, when deoxyinesine was the variable substrate. The results of alternative substrate sstudies suggested that a single enzyme acted on different nucleosides, inosine, deoxyinosine, adenosine, guanosine and deoxyguanosine.e.

• 미생물학회지
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• 제28권1호
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• pp.65-70
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• 1990

방선균의 세포분화와 관련된 연구의 일환으로 Streptomyces fradiae NRRL2702가 생성하는 단백질분해효소 저해물질을 분리정제하여 그 특성을 분석하였다. 즉 S. fradiaesm s일반 환경 조건하에서 단백질분해효소를 생성하나 특정 조건하에서는 그 단백질의 활성을 저해하는 저해제를 생성함을 알았다. 이 저해제의 분자량은 16,800이며 serine proteinasem이 일부만을 저해하는 특징이 있었다. Pronase E의 활성의 저해양상은 competitive inhibition 이었고 열에 대하여 매우 안정함을 알았다.

• Journal of Microbiology and Biotechnology
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• 제1권2호
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• pp.85-89
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• 1991

The aim of this study is to elucidate characteristics of ${\beta}-lactamase$ inhibitor produced by Streptomyces sp. SMF-19 isolated from soil was found to produce proteinaceous extracellular ${\beta}-lactamase$ inhibitor. The ${\beta}-lactamase$ inhibitor was purified through ammonium sulfate fractionation, gel filtration, anion exchange chromatography and fast performace liquid chromatography. The molecular weight of the ${\beta}-lactamase$ inhibitor was estimated to be about 48,000 by SDS-PAGE. The mode of inhibition against penicillin G as a substrate was uncompetitive. The ${\beta}-lactamase$ inhibitor was stable in wide pH range but unstable at high temperature above $50^{\circ}C$.

• 대한화학회지
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• 제14권2호
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• pp.161-168
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• 1970

In order to obtain the more effective evidence, supporting the hypothesis which have been previously described by former report that pepsin (EC 3.4. 4.1) forms a hydrophobic bond with the nonpolar side chain of its substrate, the inhibitory effect of carboxylic acids(from formic acid to iso-butyric acid) on the activity of pepsin to the synthetic dipeptide, N-Carbobenzoxy-L-glutamyl-L-tyrosine, was discussed. The kinetic study showed that the inhibition by carboxylic acids was competitive. The Kidecreased with increasing size of the inhibitor molecule. The $-{\Delta}F^{\circ}$increased linearly with increasing number of carbon atoms in the hydrocarbon chain of the inhibitor. It was confirmed that the hydrophobic bond between more than one side chain of amino acid residues(phenylalanine) in the binding region of the active center of pepsin and the side chain of amino acid residues in the substrate was formed as the first step of its enzymic mechanism. The inhibitory effect of carboxylic acids was due to the competition of the hydrocarbon group of the carboxylic acids with the side chain of the substrate for the hydrophobic binding site(the side chain of phenylalanine) of the pepsin.