A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants

Németi, Balázs; Poór, Miklós; Gregus, Zoltán

doi:10.1016/j.ab.2014.09.019

Cited by 6 publications

(14 citation statements)

References 26 publications

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“…In cell lysate, GSTO1 is inactive, presumably due to disulfide formation, but addition of a reducing agent, TCEP, activates the catalytic activity (Figure A) . We then exposed HCT‐116 cells to probe 2 at different concentrations overnight, lysed the cells, added TCEP, and measured GSTO1 activity, which revealed dose‐dependent inhibition of catalytic activity (Figure B).…”

Section: Resultsmentioning

confidence: 99%

“…[20] In cell lysate,G STO1i si nactive,p resumably due to disulfide formation, but addition of ar educing agent, TCEP, activates the catalytic activity ( Figure 4A). [21] We then exposed HCT-116 cells to probe 2 at different concentrations overnight, lysed the cells,added TCEP,and measured GSTO1 activity,which revealed dose-dependent inhibition of catalytic activity ( Figure 4B). As expected, cyclopropane 5 was inactive in this experiment ( Figure 4B)which is in accordance with the lack of labeling of GSTO1i nc ells while the optimized GSTO1i nhibitor,M L175, was active ( Figure S9).…”

Section: Angewandte Chemiementioning

confidence: 99%

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A Cyclopropene Electrophile that Targets Glutathione S‐Transferase Omega‐1 in Cells

et al. 2019

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Cyclopropenes are an important new addition to the portfolio of functional groups that can be used for bioorthogonal couplings.T he inert nature of these highly strained compounds in complex biological systems is almost counterintuitive given their established electrophilic properties in organic synthesis.H ere we provide the first demonstration of acyclopropene that is capable of direct conjugation to protein targets in cells and show that this compound preferentially alkylates the active site cysteine of glutathione S-transferase omega-1 (GSTO1).

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Section: Resultsmentioning

confidence: 99%

Section: Angewandte Chemiementioning

confidence: 99%

A Cyclopropene Electrophile that Targets Glutathione S‐Transferase Omega‐1 in Cells

et al. 2019

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“…The activity of the cytosol to reduce the GSTO1 substrate 4-NPG to 4-NAP was quantified by an HPLC-based assay . Rat liver cytosol was preincubated in sucrose buffer at 37 °C for 5 min with GSH (10 mM) in the presence or absence of a test compound.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, a major objective of the present study was to further assess the role of GSTO1 in the cytosolic reduction of DMAs V by comparing changes in the GSTO1 and DMAs V -reducing activities in response to various experimental influences. For this purpose, we have developed a novel HPLC-based GSTO1 assay, which permits quantification GSTO1 activities under conditions that are virtually identical to those of the DMAs V reductase assay, most importantly in the presence of GSH as the physiologically relevant reducing cofactor. As a specific substrate of GSTO1, S -(4-nitrophenacyl)glutathione (4-NPG; see the graphical abstract) was used in this assay.…”

Section: Introductionmentioning

confidence: 99%

Reduction of the Pentavalent Arsenical Dimethylarsinic Acid and the GSTO1 Substrate S-(4-Nitrophenacyl)glutathione by Rat Liver Cytosol: Analyzing the Role of GSTO1 in Arsenic Reduction

Németi

Poór

Gregus

2015

Chem. Res. Toxicol.

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Dimethylarsinic acid (DMAs(V)) is the major urinary metabolite of inorganic arsenic. The relatively atoxic DMAs(V) is reduced in the body to the much more toxic and thiol-reactive dimethylarsinous acid (DMAs(III)). Glutathione S-transferase omega 1 (GSTO1) can catalyze this toxification step; however, its role in the reduction of DMAs(V) in vivo or by tissue extracts is unclear. Therefore, we assessed the role of GSTO1 in the reduction of DMAs(V) to DMAs(III) by rat liver cytosol. The experiments revealed that glutathione (GSH) supported the cytosolic DMAs(V) reduction specifically and that GSH analogues and GSH conjugates, such as S-alkylglutathiones and S-(4-nitrophenacyl)glutathione (4-NPG; a GSTO1 specific substrate), inhibited the formation of DMAs(III). Observations in line with the view that GSTO1 catalyzes the cytosolic reduction of DMAs(V) include (i) findings pointing to the presence of a GSH-binding site on the DMAs(V)-reducing cytosolic enzyme, (ii) identical responsiveness of the DMAs(V)- and 4-NPG-reducing activities in rat liver cytosol to the GSTO1 specific inhibitors KT53 and chloromethylfluorescein diacetate, and (iii) perfect coelution of the two activities during affinity and anion exchange chromatography of cytosolic proteins. Other observations appear ambiguous as to the role of GSTO1 in the cytosolic reduction of DMAs(V). These include the different sensitivities of the DMAs(V)-reducing and GSTO1 activities to aurothioglucose, trivalent antimony, and zinc ions, as well as the preserved GSTO1 activity in cytosols whose DMAs(V)-reducing activity was lost due to spontaneous thiol oxidation. These disparate findings may be reconciled by assuming that GSTO1 catalyzes the reduction of both DMAs(V) and 4-NPG in rat liver cytosol; however, the enzyme employs different sites and/or mechanisms when reducing these substrates.

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“…Towards this end, we exploited the known specific activity of GSTO1t of unction as aS -(phenacyl)-glutathione reductase and prepared S-(4-Nitrophenacyl)glutathione (4-NPG) whose conversion to 4-nitroacetophenone can be specifically measured by absorbance at 305 nm ( Figure 4A). [21] We then exposed HCT-116 cells to probe 2 at different concentrations overnight, lysed the cells,added TCEP,and measured GSTO1 activity,which revealed dose-dependent inhibition of catalytic activity ( Figure 4B). [21] We then exposed HCT-116 cells to probe 2 at different concentrations overnight, lysed the cells,added TCEP,and measured GSTO1 activity,which revealed dose-dependent inhibition of catalytic activity ( Figure 4B).…”

Section: Angewandte Chemiementioning

confidence: 99%

A Cyclopropene Electrophile that Targets Glutathione S‐Transferase Omega‐1 in Cells

et al. 2019

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show abstract

A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants

Cited by 6 publications

References 26 publications

A Cyclopropene Electrophile that Targets Glutathione S‐Transferase Omega‐1 in Cells

A Cyclopropene Electrophile that Targets Glutathione S‐Transferase Omega‐1 in Cells

Reduction of the Pentavalent Arsenical Dimethylarsinic Acid and the GSTO1 Substrate S-(4-Nitrophenacyl)glutathione by Rat Liver Cytosol: Analyzing the Role of GSTO1 in Arsenic Reduction

A Cyclopropene Electrophile that Targets Glutathione S‐Transferase Omega‐1 in Cells

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