• Biotechnology and Bioprocess Engineering:BBE
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• 제9권1호
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• pp.23-34
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• 2004

Escherichia coli transcription termination factor Rho catalyzes the unwinding of RNA/DNA duplex in reactions that are coupled to ATP binding and hydrolysis. Fluorescence stopped-flow methods using ATP and the fluorescent 2'(3')-O-( N-methylanthraniloyl) derivatives (mant-derivatives) of ATP and ADP were used to probe the kinetics of nucleotide binding to and dissociation from the Rho-RNA complex. Presteady state nucleotide binding kinetics provides evidence for the presence of negative cooperativity in nucleotide binding among the multiple nucleotide binding sites on Rho hexamer. The binding of the first nucleotide to the Rho-RNA complex occurs at a bimolecular rate of 3.6${\times}$10$\^$6/ M$\^$-1/ sec$\^$-1/ whereas the second nucleotide binds at a slower rate of 4.7${\times}$10$\^$5/ M$\^$-1/ sec$\^$-1/ at 18$^{\circ}C$, RNA complexed with Rho affects the kinetics of nucleotide interaction with the active sites through conformational changes to the Rho hexamer, allowing the incoming nucleotide to be more accessible to the sites. Adenine nucleotide binding and dissociation is more favorable when RNA is bound to Rho, whereas ATP binding and dissociation step in the absence of RNA occurs significantly slower, at a rate ∼70- and ∼40-fold slower than those observed with the Rho-RNA complex, respectively.

• Journal of Microbiology and Biotechnology
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• 제17권7호
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• pp.1191-1197
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• 2007

The detection and kinetics of mucosal pathogenic bacteria binding on polysaccharide ligands were studied using a surface plasmon resonance biosensor. The kinetic model applied curve-fitting to the experimental surface plasmon resonance sensorgrams to evaluate the binding interactions. The kinetic parameters for the mucosal pathogenic bacteria (Pseudomonas aeruginosa, Pseudomonas fluorescens, Serratia marcescens) with the alginate ligand were determined from a kinetic model. In addition, the binding interactions of the mucosal pathogenic bacteria with polysaccharide binding pairs (Pseudomonas aeruginosa/alginate, Streptococcus pneumoniae/pneumococcal polysaccharide, Staphylococcus aureus/pectin) were also compared with their kinetic parameters. The rate constants of association for Pseudomonas aeruginosa with the alginate ligand were higher than those for Pseudomonas fluorescens. Serratia marcescens had no detectable interaction with the alginate ligand. The adhesion affinity of Pseudomonas aeruginosa with alginate was higher than that for the other binding pairs. The binding affinities of the pathogenic bacteria with their own polysaccharide were higher than that of Staphylococcus aureus with pectin. Measuring the contact angle was found to be a feasible method for detecting binding interactions between analytes and ligands.

• Bulletin of the Korean Chemical Society
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• 제27권2호
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• pp.224-230
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• 2006

Escherichia coli transcription termination factor Rho catalyzes the unwinding of RNA/DNA duplex in reactions that are coupled to ATP binding and hydrolysis. We report here the kinetic mechanism of presteady state ATP binding and hydrolysis by the Rho-RNA complex. Presteady state chemical quenched-flow technique under multiple turnover condition was used to probe the kinetics of ATP binding and hydrolysis by the Rho-RNA complex. The quenched-flow presteady state kinetics of ATP hydrolysis studies show that three ATPs are bound to the Rho-RNA complex with a rate of $4.4\;{\times}\;10^5M^{-1}s^{-1}$, which are subsequently hydrolyzed at a rate of $88s^{-1}$ and released during the initiation process. Global fit of the presteady state ATP hydrolysis kinetic data suggests that a rapid-equilibrium binding of ATP to Rho-RNA complex occurs prior to the first turnover and the chemistry step is not reversible. The initial burst of three ATPs hydrolysis was proposed to be involved in the initialization step that accompanies proper complex formation of Rho-RNA. Based on these results a kinetic model for initiation process for Rho-RNA complex was proposed relating the mechanism of ATP binding and hydrolysis by Rho to the structural transitions of Rho-RNA complex to reach the steady state phase, which is implicated during translocation along the RNA.

• Journal of Microbiology
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• 제33권4호
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• pp.289-294
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• 1995

The action mechanism of hemolysin rendering virulency of Vibrio anguilarum has not clarified as yet, even though there were several possible factors explained. We have studied hemolytic kinetics performed by hemolysin from V. anguillarum strain V7 as well as binding of hemolysin to RBC membrane. Maximal rate of hemolysis and duration of lag phase were directly and inversly correlated to the concentration of hemolysin used. Hemolysin molecules are known to bind consumptively with proper diameter, while other protectants with smaller diameter could not. In conclusion, hemolysin should bind irreversibly to RBC membrane exert hemolysis distorting osmotic pressure. The binding could be hindered by spatial structure of the RBC surfacem which might be caused by sialic acid.

• 대한화학회지
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• 제14권2호
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• pp.155-160
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• 1970

The kinetics of the pepsin-catalyzed hydrolysis of N-carbobenzoxy-L-glutamyl-L-tyrosine at pH 3.5 and $37^{\circ}C$ were determined by a spectrophotometric technique. The pepsin used was further purified on a Sephadex G-75 column. The kinetics data were Km = l.7 ${\times}10^{-3}M,\;-{\Delta}F^{\circ}$ = 3.99Kcal/mole, and $k^3=\;2.1{\times}10^{-2}\;sec^{-1}$. An analysis of the above data and other investigators' data obtained from some dipeptides led to the following conclusions. (1) Phenylalanyl residues in a synthetic peptide are bound to pepsin more strongly than glutamyl or tyrosyl residues, supporting the theory that a part of the binding region of the active center is hydrophobic. (2) Dipeptides are bound to pepsin principally through their side chains and the binding involves both side-chain residues. (3) The nature of amino acids in dipeptides $R_2-R_1,\;affect\;the\;k_3$ values.

• Bulletin of the Korean Chemical Society
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• 제25권5호
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• pp.726-736
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• 2004

In this paper, kinetics of reaction between Bromophenol blue (BPB) and $OH^-$, called fading, has been studied through a spectrophotometric method in the presence of nonionic Triton X-100 (TX-100), anionic sodium dodecyl sulfate (SDS) and cationic dodecyl trimethylammonium bromide (DTAB) surfactants. The influence of changes in the surfactant concentration on the observed rate constant was investigated. The results are treated quantitatively by pseudophase ion-exchange (PPIE) model and a new simple model called "classical model". The binding constants of BPB molecules to the micelles and free molecules of surfactants, their stoichiometric ratios and thermodynamic parameters of binding have been evaluated. It was found that SDS has nearly no effect on the fading rate up to 10 mM, whereas TX-100 and DTAB interact with BPB which reduce the reaction rate. By the use of fading reaction of BPB, the binding constants of SDS molecules to TX-100 micelles and their Langmuir and Freundlich adsorption isotherms were obtained and when mixtures of DTAB and TX-100 were used, no interaction was observed between these two surfactants.

• BMB Reports
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• 제34권3호
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• pp.194-200
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• 2001

Secreted arginase from Evernia prunastri thallus has been purified 616-fold from the incubation medium. Purified arginase was resolved as only one peak in a capillary electrophoresis with a pI value of 5.35. The protein contained high amounts of acidic amino acids, such as Asx and Glx, and a relatively high quantity of Ser and Gly. The molecular mass of native, purified arginase was estimated as about 26 kDa by SE-HPLC. Substrate saturated kinetic showed a typical Michaelis-Menten relationship with a K_m value of 3.3 mM L-arginine. Atranorin behaved as a mixed activator of the enzyme (apparent $K_m$ = 0.96 mM); whereas evernic and usnic acid were revealed as non competitive inhibitors (apparent $K_m$ values were 3.16 mM and 3.05 mM, respectively). Kinetics of atranorin binding indicated that saturation was reached from 0.18 ${\mu}mol$ of the total atranorin and the occurrence of multiple sites for the ligand. This agrees with a possible aggregation of several enzyme subunits during the interaction process. A value of binding sites of about 12 was obtained. The binding of evernic acid was saturated from 23 nmol of total phenol. The number of binding sites was about 5. The loss of the binding ability of evernic acid could be interpreted as a single negative cooperatively. Usnic acid behaves in a similar way to evernic acid, although the binding saturation occurs at $0.14\;{\mu}moles$ of the ligand. This binding appears to be unspecific, and has 28 usnic acid binding sites to the protein.

• Bulletin of the Korean Chemical Society
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• 제3권3호
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• pp.122-129
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• 1982

In order to extend our understanding on the multiple inhibition enzyme kinetics, a general equation of an enzyme reaction in the presence of two different reversible inhibitors was derived by what we call "match-box mechanism" under the combined assumption of steady-state and quasi-equilibrium for inhibitor binding. Graphical methods were proposed to analyze the multiple inhibition of an enzyme by any given sets of different inhibitors, i.e., competitive, noncompetitive, and uncompetitive inhibitors. This method not only gives an interaction factor $({\alpha})$ between two inhibitors, but also discerns ${\alpha}_1$ and ${\alpha}_2$ with and without substrate binding, respectively. The factors involved in the dissociation constants of inhibitors can also be evaluated by the present plot. It is also shown that the present kinetic approach can be extended to other forms of activators or hydrogen ions with some modification.

• 한국미생물·생명공학회지
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• 제47권1호
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• pp.96-104
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• 2019

Beauveria bassiana, one of the most common entomopathogenic fungi, has been isolated, pre defined and characterized in-house from soil of tea cultivation area. Experiments have been performed to verify the presence of chitinase as intracellular metabolite and its release as extracellular product rendering the spores with biopesticide activity. Although there are many responsible enzymes for the pest killer action of B. bassiana, binding property of chitinase depending on presence as well as absence of serine supplemented in the media has been studied with respect to the production and kinetics. A programmed investigation conclusively indicates that the isolated spore (hyphae) of B. bassiana has been metabolically enriched with the enzyme chitinase in presence of an externally added amino acid serine with its inhibitory kinetics.

• Bulletin of the Korean Chemical Society
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• 제30권9호
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• pp.2051-2056
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• 2009

The rate constants of alkaline fading of malachite green ($MG^+$) was measured in the presence of nonionic (TX-100), cationic (DTAB) and anionic (SDS) surfactants. This reaction was studied under pseudo-first-order conditions at 283∼303 K. The rate of fading reaction showed noticeable dependence on the electrical charge of the used surfactants. It was observed that the reaction rate constants were increased in the presence of TX-100 and DTAB and decreased in the presence of SDS. According to Hughs-Ingold rules for nucleophilic substitution reactions, the electric charge of MG/surfactant compound along with decrease in dielectric constant of $MG^+$ micro-environment in this compound varies the rate of fading reaction. Binding constants of surfactant molecules to $MG^+$ were calculated using cooperativity, pseudo-phase ion exchange and classical models and the related thermodynamic parameters were obtained by classical model. The results show that the binding of $MG^+$ to TX-100 is exothermic and binding of $MG^+$ to DTAB and SDS in some concentration ranges of the used surfactants is endothermic and in the other ones is exothermic.