• BMB Reports
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• v.36 no.2
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• pp.149-153
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• 2003

The dibucaine number (DN) was determined for serum cholinesterase (EC 3.1.1.8, SChE) in plasma samples. The ones with a DN of 79-82 were used, because they had the "usual" SChE variant. The enzyme was assayed colorimetrically by the reaction of 5,5'-dithiobis-[2-nitrobenzoic acid] (DTNB) with the free sulfhydryl groups of thiocholine that were produced by the enzyme reaction with butrylthiocholine (BuTch) or acetylthiocholine (AcTch) substrates, and measured at 412 nm. Dibucaine, a quaternary ammonium compound, inhibited SChE to a minimum within 2 min in a reversible manner. The inhibition was very potent. It had an $IC_{50}$ of $5.3\;{\mu}M$ with BuTch or $3.8\;{\mu}M$ with AcTch. The inhibition was competitive with respect to BuTch with a $K_i$ of $1.3\;{\mu}M$ and a linear-mixed type (competitive/noncompetitive) with respect to AcTch with inhibition constants, $K_i$ and $K_I$ of 0.66 and $2.5\;{\mu}M$, respectively. Dibucaine possesses a butoxy side chain that is similar to the butryl group of BuTch and longer by an ethylene group from AcTch. This may account for the difference in inhibition behavior. It may also suggest the existence of an additional binding site, other than the anionic binding site, and of a hydrophobic nature.

• Journal of the Korean Society of Food Science and Nutrition
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• v.9 no.1
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• pp.59-73
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• 1980

A detailed procedure was described for the isolation of cratine kinase (ATP-Creatine phosphotransferase, E. C. 2. 7. 3. 2.) from the muscle of the snake Bungarus fasciatus. The original isolation procedure of Kuby et al. for the rabbit muscle enzyme has been modified and extended to include a chromatographic step. The properties of the enzyme have been investigated and kinetic constants for the reverse reactions determined as the followings: 1) A molecular weight of the enzyme was determined by gel filteration on Sephadex G-100 and by electrophoresis on SDS-polyacrylamide was 86,000. 2) Two reactive sulphydryl groups were detected with dithiobis nitrobenzoic acid (DTNB). 3) The nucleotide substrate specificity in the reverse reaction was determined as ADP*2'-dADP＞GDP＞XDP＞UDP with magnesium as the activating metal ion. 4) The order of the metal specificity in the reverse reaction Mg>Mn>$Ca{\sim}Co$ was determined with ADP as substrate. 5) A detailed kinetic analysis was carried out in the reverse direction with $MaADP^-$ as the nucleotide substrate. Initial velocity and product inhibition studies($MaADP^{2-}$ competitive with respect to MgADP- and noncompetitive with respect to $N-phosphorycreatine^{2-}$ ; Creatine competitive with respect to $N-phosphorycreatine^{2-}$ and noncompetitive with respect to Ma $ADP^-)$ indicated that the reaction obeyed a sequential mechanism of the rapid equilibrium random type.

• BMB Reports
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• v.35 no.2
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• pp.178-185
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• 2002

The Arabidopsis thaliana S-Adenosylmethionine decarboxylase (AdoMetDC) cDNA ($GenBank^{TM}$ U63633) was cloned. Site-specific mutagenesis was performed to introduce mutations at the conserved cysteine $Cys^{50}$, $Cys^{83}$, and $Cys^{230}$, and $lys^{81}$ residues. In accordance with the human AdoMetDC, the C50A and C230A mutagenesis had minimal effect on catalytic activity, which was further supported by DTNB-mediated inactivation and reactivation. However, unlike the human AdoMetDC, the $Cys^{50}$ and $Cys^{230}$ mutants were much more thermally unstable than the wild type and other mutant AdoMetDC, suggesting the structural significance of cysteines. Furthermore, according to a circular dichroism spectrum analysis, the $Cys^{50}$ and $Cys^{230}$ mutants show a higher a-helix content and lower coiled-coil content when compared to that of wild type and the other mutant AdoMetDC. Also, the three-dimensional structure of Arabidopsis thaliana AdoMetDC could further support all of the data presented here. Summarily, we suggest that the $Cys^{50}$ and $Cys^{230}$ residues are structurally important.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.04a
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• pp.157-167
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• 2000

A thermostable chitosanase gene from the isolated strain, Bacillus sp. CK4, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30 kDa enzyme in size. The deduced amino acid sequence of the chitosanase from Bacillus sp. CK4 exhibits 76.6%, 15.3%, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. CK4 belongs to the Cluster III group with Bacillus subtilis. The size of the gene was similar to that of a mesophile, Bacillus subtilis showing a higher preference for codons ending in G or C. The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues were changed to E50D/Q, E62D/Q, and D66N/E by site-directed mutagenesis. The D66N/E mutants enzymes had remarkably decreased kinetic parameters such as $V_{max}$ and k$\sub$cat/, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three cysteine residues at position 49, 72, and 211. Titration of the Cys residues with DTNB showed that none of them were involved in disulfide bond. The C49S and C72S mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However the half-life of the C211S mutant enzyme was less than 60 min at 80$^{\circ}C$, while that of the wild type enzyme was about 90 min. Moreover, the residual activity of C211S was substantially decreased by 8 M urea, and fully lost catalytic activity by 40% ethanol. These results show that the substitution of Cys with Ser at position 211 seems to affect the conformational stability of the chitosanase.

• International Journal of Oral Biology
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• v.33 no.3
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• pp.117-123
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• 2008

Reactive oxygen species (ROS) are toxic agents that may be involved in various neurodegenerative diseases. Recent studies indicate that ROS can act as modulators of neuronal activity, and are critically involved in persistent pain primarily through spinal mechanisms. In the present study, whole cell patch clamp recordings were carried out to investigate the effects of tert-buthyl hydroperoxide (t-BuOOH), an ROS, on neuronal excitability and the mechanisms underlying changes of membrane excitability. In current clamp condition, application of t-BuOOH caused a reversible membrane depolarization and firing activity in substantia gelatinosa (SG) neurons. When slices were pretreated with phenyl-N-tert-buthylnitrone (PBN) and ascorbate, ROS scavengers, t-BuOOH failed to induce membrane depolarization. However, isoascorbate did not prevent t-BuOOH-induced depolarization, suggesting that the site of ROS action is intracellular. The t-BuOOH-induced depolarization was not blocked by pretreatment with dithiothreitol (DTT), a sulfhydryl-reducing agent. The membrane-impermeant thiol oxidant 5,5-dithiobis 2-nitrobenzoic acid (DTNB) failed to induce membrane depolarization, suggesting that the changes of neuronal excitability by t-BuOOH are not caused by the modification of extrathiol group. The t-BuOOH-induced depolarization was suppressed by the phospholipase C (PLC) blocker U-73122 and inositol triphosphate ($IP_3$) receptor antagonist 2-aminoethoxydiphenylbolate (APB), and after depletion of intracellular $Ca^{2+}$ pool by thapsigargin. These data suggest that ROS generated by peripheral nerve injury can induce central sensitization in spinal cord, and t-BuOOH-induced depolarization may be regulated by intracellular $Ca^{2+}$ store mainly via $PLC-IP_3$ pathway.

• BMB Reports
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• v.41 no.1
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• pp.62-67
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• 2008

The present study was aimed to elucidate the mechanism of stabilization of tubulin by deuterium oxide ($D_2O$). Rate of decrease of tryptophan fluorescence during aging of tubulin at 4$^{\circ}C$ and 37$^{\circ}C$ was significantly lower in $D_2O$ than in $H_2O$. Circular dichroism spectra of tubulin after incubation at 4$^{\circ}C$, suggested that complete stabilization of the secondary structure in D2O during the first 24 hours of incubation. The number of available cysteine measured by DTNB reaction was decreased to a lesser extent in $D_2O$ than in $H_2O$. . During the increase in temperature of tubulin, the rate of decrease of fluorescence at 335 nm and change of CD value at 222 nm was lesser in $D_2O$. Differential Scanning calorimetric experiments showed that the $T_m$ values for tubulin unfolding in $D_2O$ were 58.6$^{\circ}C$ and 62.17$^{\circ}C$, and in $H_2O$. those values were 55.4$^{\circ}C$ and 59.35$^{\circ}C$.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.17
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• pp.7129-7135
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• 2014

Background: In this study, we aimed to evaluate the growth inhibitory effect of the combination of ethaselen (BBSKE) and low fixed dose of selenite against A549 human non-small cell lung cancer cells in vitro. Materials and Methods: Growth inhibitory effects against A549 cells were determined by SRB assay. Combination index (CI) values were calculated based on Chou-Talalay median-effect analyses. Dose reduction index (DRI) values were applied to calculate dose reduction of selenite. Contents of free thiols and GSH were determined by DTNB assay and intracellular ROS levels by DCFH-DA fluorescence labeling. Results: Compared with BBSKE or selenite single treatment, the combined application of ethaselen and a low fixed dose of selenite shortened the onset time of sodium selenite, reduced $IC_{50}$ values, and increased the maximum inhibition rates, suggesting a possible molecular mechanism of the synergism. Obvious synergistic effects were observed after different times of combination treatment, especially after 24 h. Compared with selenite single treatment, dosage of selenite could be remarkably reduced in combination therapy to gain the same inhibitory effect on cell proliferation. Compared with BBSKE single treatment, the content of free thiols and GSH were significantly reduced and ROS levels greatly elevated in the combination group. For the combination treatment, cell viability increased as greater concentrations of GSH were added. Conclusions: All these results indicate that the combination treatment of BBSKE and selenite showed synergism to inhibit A549 cell proliferation in vitro, and also reduced the selenite dosage to mitigate its toxicity which is very meaningful for combination chemotherapy of lung cancer. The synergism was probably caused by the accelerated exhaustion of intracellular reductive substances, such as free thiols and GSH, which ultimately leads to enhanced oxidative stress and apoptosis.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.9
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• pp.5421-5425
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• 2013

Background: To assess the antioxidant effects of gamma-oryzanol on human prostate cancer cells. Materials and Methods: Cytotoxic activity of gamma-oryzanol on human DU145 and PC3 prostate cancer cells was determined by proliferation assay using 3-(4, 5-dimethylthiazol, 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) reagent. mRNA levels of genes involved in the intracellular antioxidant system, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GSR) were determined by reverse transcription-polymerase chain reaction (RT-PCR). Cancer cell lysates were used to measure lipid peroxidation using thiobarbituric acid reactive substance (TBARS). Glutathione contents of the cell lysates were estimated by the reaction between sulfhydryl group of 5, 5'-dithio (bis) nitrobenzoic acid (DTNB) to produce a yellow-color of 5-thio-2-nitrobenzoic acid using colorimetric assay. Catalase activity was also analysed by examining peroxidative function. Protein concentration was estimated by Bradford's assay. Results: All concentrations of gamma-oryzanol, 0.1-2.0mg/ml, significantly inhibited cell growth in a dose- and time-dependent fashion in both prostate cancer cell lines, DU145 and PC3. Gene expression of catalase in DU145 and PC3 exposed to gamma-orizanol at 0.5mg/ml for 14 days was down regulated, while mRNA of GPX was also down regulated in PC3. The MDA and glutathione levels including catalase activity in the cell lysates of DU145 and PC3 treated with gamma-oryzanol 0.1 and 0.5mg/ml were generally decreased. Conclusions: This study highlighted effects of gamma-oryzanol via the down-regulation of antioxidant genes, catalase and GPX, not cytotoxic roles. This might be interesting for adjuvant chemotherapy to make prostate cancer cells more sensitive to free radicals. It might be useful for the reduction of cytotoxic agents and cancer chemoprevention.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.235-242
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• 1999

We investigated the growth-inhibitory mechanism of Helicobacter pylori by omeprazole (OMP) and its activated sulfenamide (OAS). Using dithiothreitol (DTT) and 5,5'-dithio-bis[2-nitrobenzoic acid] (DTNB; Ellman's reagent), we first determined the relationship between the binding capacity of these compounds to H. pylori membrane and its significance to membrane P-type ATPase activity. After incubation of the intact H. pylori cells with either OMP or OAS, the residual quantity of free SH-groups on the cell membrane was measured, and, the resulting values were plotted as a function of time. From this experiment, we found that there was a considerable difference in the membrane-binding rates between OMP and OAS. At neutral pH, the disulfide bond formation on H. pylori membrane was completed within 2 min of incubation of the intact cells with OAS. By OMP, however, it was gradually formed, exceeding 10 min of incubation for completion, whereby, the extent of P-type ATPase inhibition appeared to be proportional to the disulfide forming rate. From this data, it was suggested that the disulfide formation might directly affect enzyme activity. Since OMP per se cannot yield a disulfide bond with cysteine, it is predicted that the enzyme inactivation must be caused by the OAS form. Accordingly, we postulated that, under the neutral pH, OMP could be converted to OAS in the course of transport. By extrapolating the inhibitory slopes, we could evaluate K₁ values, relating to their minimal inhibitory concentrations (MICs) for H. pylori growth. In these MIC ranges, H. pylori uptake or vesicular export of nutrients such as peptides were totally prohibited, but their effect in Escherichia coli were negligible. From these observations, we strongly suggest that the P-type ATPase activity is essential for the survival of H. pylori cells in particular.

• Animal cells and systems
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• v.15 no.2
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• pp.131-138
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• 2011

Redox regulation is one of the ubiquitous mechanisms to modulate ion channels. We here investigated how 5,5'-dithio-bis (2-nitrobenzoic acid), a cysteine specific oxidizing reagent, modulates $Ca_v3.1$ and $Ca_v3.2$ T-type $Ca^{2+}$ channels expressed in Xenopus oocytes. Application of the reagent inhibited $Ca_v3.1$ and $Ca_v3.2$ currents in a dose-dependent manner. The oxidizing reagent (1 mM) reduced the peak amplitude of $Ca_v3.1$ and $Ca_v3.2$ currents by ~50% over 2-3 minutes and the decreased currents were fully recovered upon washout of it. The reagent slowed the activation and inactivation kinetics of $Ca_v3.1$, $Ca_v3.2$, and $Ca_v3.3$ channel currents. Notably, the reagent positively shifted both activation and steady-state inactivation curves of $Ca_v3.1$, while it did not those of $Ca_v3.2$. Utilizing chimeric channels from $Ca_v3.1$ and $Ca_v3.2$, we localized the domains III and IV of $Ca_v3.1$ responsible for the positive shifts of channel activation and steady-state inactivation. These findings provide hints relevant to the electrophysiological and molecular mechanisms accounting for the oxidative regulation of T-type channels.