• Title/Summary/Keyword: 5,5'-dithio-bis-(2-nitrobenzoic acid)

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S-Hippuryl Thioglycolyl Glycine : A New Chromogenic Substrate for Dipeptidyl Carboxypeptidases (Dipeptidyl Carboxypeptidases에 의한 S-Hippuryl Thioglycolyl Glycine의 가수분해)

  • Lee Hyun-Jae
    • Journal of the Korean Chemical Society
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    • v.19 no.4
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    • pp.246-251
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    • 1975
  • A spectrophotometric assay technique is descriead for the measurement of free SH-groups in the enzyme reaction mixture. The method utilizes a new substrate, S-hippuryl-thioglycolyl-glycine(S-Hip-thioglycol-Gly) which is the basis for a convenient assay of angiotensin-converting enzyme and other dipeptidyl carboxypeptidases. This substrate contains an appropriately located thioester linkage that is hydrolyzed by the converting enzyme and other dipeptidyl carboxypeptidases. One of the products, thioglycolyl glycine, is readily measured by reaction with Ellman's reagent, 5,5'-dithio-bis-(2-nitrobenzoic acid), DTNB, to produce 5-thio-2-nitrobenzoic acid which has a strong absorption band at 410 nm. The method is sensitive (${\varepsilon}M = 1.36{\times}10^4$ at 412 nm) and can be applied as a continuous recording with DTNB present in the enzymatic reaction mixture.

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Functional Amino Acid Residues of Recombinant Tobacco Acetolactate Synthase

  • Chong, Chom-Kyu;Chang, Soo-Ik;Choi, Jung-Do
    • BMB Reports
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    • v.31 no.3
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    • pp.258-263
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    • 1998
  • Acetolactate synthase (ALS) is the common enzyme in the biosynthetic pathways leading to leucine, valine, and isoleucine. Tobacco ALS was expressed in E. coli and purified to homogeneity. The recombinant tobacco ALS was inactivated by thiol-specific reagents, N-ethylmaleimide (NEM) and 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). Inactivation of the ALS by NEM followed pseudo-first order kinetics and was first order with respect to the modifier. The substrate pyruvate protected the enzyme against the inactivation by NEM and DTNB. Extrapolation to complete inactivation of the enzyme by DTNB showed modification of approximately 2 out of 4 total cysteinyl residues (or 2 cysteinyl and 1 cysteinyl residues), with approximately 1 residue protected by pyruvate. The tobacco ALS was also inactivated by the tryptophanspecific reagent, N-bromosuccinimide (NBS), and was similarly protected by pyruvate. The kinetics of the inactivation was first-order with respect to NBS. The present data suggest that cysteinyl and tryptophanyl residues play a key role in the catalytic function of the enzyme.

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The Inhibition of TREK2 Channel by an Oxidizing Agent, 5,5'-dithiobis (2-nitrobenzoic acid), via Interaction with the C-terminus Distal to the 353rd Amino Acid

  • Park, Kyoung-Sun;Bang, Hyo-Weon;Shin, Eun-Young;Kim, Chan-Hyung;Kim, Yang-Mi
    • The Korean Journal of Physiology and Pharmacology
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    • v.12 no.4
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    • pp.211-216
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    • 2008
  • TREK (TWIK-RElated $K^+$ channels) and TRAAK (TWIK-Related Arachidonic acid Activated $K^+$ channels) were expressed in COS-7 cells, and the channel activities were recorded from inside-out membrane patches using holding potential of - 40 mV in symmetrical 150 mM $K^+$ solution. Intracellular application of an oxidizing agent, 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB), markedly decreased the activity of the TREK2, and the activity was partially reversed by the reducing agent, dithiothreitol (DTT). In order to examine the possibility that the target sites for the oxidizing agents might be located in the C-terminus of TREK2, two chimeras were constructed: TREK2 (1-383)/TASK3C and TREK2 (1-353)/TASK3C. The channel activity in the TREK2 (1-383)/TASK3C chimera was still inhibited by DTNB, but not in the TREK2 (1-353)/TASK3C chimera. These results indicate that TREK2 is inhibited by oxidation, and that the target site for oxidation is located between the amino acid residues 353 and 383 in the C-terminus of the TREK2 protein.

DTNB oxidation effects on T-type $Ca^{2+}$ channel isoforms

  • Lee, Sang-Soo;Kang, Ho-Won;Park, Jin-Yong;Lee, Jung-Ha
    • 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.

Antioxidant Effects of Gamma-oryzanol on Human Prostate Cancer Cells

  • Klongpityapong, Papavadee;Supabphol, Roongtawan;Supabphol, Athikom
    • 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.

Biochemical Characteristics of a Palmitoyl Acyl Carrier Protein Thioesterase Purified from Iris pseudoacorus

  • Kang, Han-Chul;Hwang, Young-Soo
    • BMB Reports
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    • v.29 no.5
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    • pp.436-441
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    • 1996
  • The palmitoyl acyl carrier protein (ACP) specific thioesterase (EC 3.1.2.14) from Iris pseudoacorus was purified and characterized. The thioesterase which was very unstable in relatively high salt concentrations was eluted using a co-gradient of Triton X-100 and low concentration of KCl or Na-phosphate from Q-Sepharose, DEAE-Sepharose, and hydroxyapatite chromatography. SDS-PAGE analysis showed a single band with a molecular weight of 35,000. The native molecular weight of approximately 37,000 was estimated by Sephacryl S-200 chromatography, indicating that the enzyme is a monomer. The thioesterase activity was inhibited about 75% and 50% by N-ethylmaleimide (2 mM) and phenylmethylsulfonyl fluoride (2 mM). respectively. The N-ethylmaleimide-inactivation was protected by sodium palmitate but the inactivation with phenylmethylsulfonyl fluoride was not protected. Oxidation of thiols by 2 mM 5.5'-dithio-bis-(2-nitrobenzoic acid) resulted in 65% inactivation of the enzyme. These results suggest that a cysteinyl residue is essential to the catalytic reaction of the enzyme. The enzyme activity was increased by sodium citrate and also by $Cu^{2+}$

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Selective Homocysteine Assay with Cucurbit[7]uril by pH Regulation

  • Bae, Won-Bin;Kim, Hee-Joon;Jhee, Kwang-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.32 no.4
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    • pp.514-521
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    • 2022
  • We report the effect of pH on the supramolecular complexation of two biothiols, viz., homocysteine (Hcy) and cysteine (Cys), with cucurbit[7]uril (CB[7]). Under basic pH conditions, Cys did not complex with CB[7], whereas Hcy efficiently complexed with CB[7], as confirmed by 1H NMR spectroscopy and Ellman's reagent (5,5'-dithio-bis(2-nitrobenzoic acid), DTNB) assay. 1H NMR and Raman spectroscopic studies revealed that, in the absence of CB[7], Hcy auto-oxidized slowly (~36 h) to homocystine (HSSH) under basic pH conditions. However, the rate of Hcy oxidation increased by up to 150 fold in the presence of CB[7], as suggested by the DTNB assay. Thus, supramolecular complexation under basic pH conditions led to the formation of a HSSH-CB[7] complex, and not Hcy-CB[7]. The results indicate that Hcy is rapidly oxidized to HSSH under the catalysis of CB[7], which acts as a reaction chamber, in basic pH conditions. Our studies suggest that Hcy concentration, a risk factor for cardiovascular disease, can be selectively and more easily quantified by supramolecular complexation with CB [7].

Effect of Omeprazole on Membrane P-Type ATPase and Peptide Transport in Helicobacter pylori

  • KI, MI-RAN;SOON-KYU YUN;SE-YOUNG HWANG
    • 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.

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