Glutathione <i>S</i>‐Conjugate Formation from 1‐Chloro‐2,4‐dinitrobenzene and Biliary <i>S</i>‐Conjugate Excretion in the Perfused Rat Liver

Wahlländer, A.; Sies, Helmut

doi:10.1111/j.1432-1033.1979.tb13056.x

Cited by 100 publications

(44 citation statements)

References 17 publications

(10 reference statements)

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“…As shown, the mBCI-GSH conjugation during the first 8 min initially follows a first-order kinetic reaction. However, the conjugation reaction will not reach a plateau due to the second-order kinetic reaction caused by a number of possible additional reactions [e.g., conjugation of mBCl to nonprotein thiols (21), new synthesis of GSH (3,17,34), feedback inhibition by mBC1-GSH on GST activity (9), slow leakage of the mBC1-GSII conjugate from cells (32)]. Curve-fitting regression analysis was performed using scans that follow the first-order kinetic reaction to minimize the confounding factors referenced above ( i c , where the correlation coefficient = 1 ).…”

Section: Analysis Of Gsh-mbci Fluorescencementioning

confidence: 99%

Kinetic analysis of glutathione in anchored cells with monochlorobimane

1995

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A method for the measurement of intracellular glutathione content and glutathione S-transferase activity with monochlorobimane in adherent cells is described. The method involves the kinetic analysis of monochlorobimane conjugation to glutathione over a relatively short period of time. This permits extrapolation over time for determination of equilibrium fluorescence intensity (relative glutathione level) ftom scan intensity data that follows first-order kinetics, minimizing problems commonly associated with the use of monochlorobimane. By using measured fluorescence intensity values from glutathione standards, a suspension calibration curve was generated and, subsequently, was used to determine the photomultiplier tube saturation rate. A theoretical intracellular calibration curve was then generated to quantify glutathione content in cells. This method was also applied to study the changes in glutathione in a variety of rodent and human cell lines and in selected cocultures of cells exhibiting similar or different glutathione levels. Comparison of the glutathione levels obtained with monochlorobimane and a standard colorimetric method (GSH-400) indicated good correlation between the two methods. These studies support the use of laser cytometry for measuring intracellular glutathione with monochlorobimane as well as changes in glutathione occurring in cells that establish physical contacts with other cells. Laser cytometric analysis of glutathione in anchored cells also provides opportunities to monitor individual cellular responses to a variety of experimental manipulations, such as responses to various toxic insults or the protective effects of gap junction-mediated intercellular communication. o 1995 wiley-Liss, I~C .

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Section: Analysis Of Gsh-mbci Fluorescencementioning

confidence: 99%

Kinetic analysis of glutathione in anchored cells with monochlorobimane

1995

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“…The intracellular formation of glutathione Sconjugates is followed by their active extrusion from the cell [3-51. In liver, glutathione Sconjugates and glutathione disulfide (GSSG) are secreted preferentially into bile [4,6] by, what appears to be, a common transport system [7]. A similar transport system, involved in the extrusion * To whom correspondence should be addressed…”

Section: Introductionmentioning

confidence: 99%

Glutathione S‐conjugates stimulate ATP hydrolysis in the plasma membrane fraction of rat hepatocytes

Nicotera¹,

Baldi²,

Svensson³

et al. 1985

FEBS Letters

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Incubation of a rat hepatocyte plasma membrane fraction with micromolar concentrations of either glutathione disultide or various glutathione S-conjugates resulted in a several-fold increase in the rate of ATP hydrolysis. This stimulation was further enhanced when the plasma membrane fraction had been pretreated with agents that arylate or oxidize sulthydryl groups, suggesting that this ATPase activity is modulated by the protein thiol status of the plasma membrane. It is proposed that this newly discovered ATPase may function in the cellular extrusion of both glutathione disultide and glutathione s-conjugates. HepatocytePlasma membrane

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“…In situ, CDNB is conjugated with GSH at a high rate to give DNPG [16]. Consequently, the interactions of this conjugate with the enzyme GSSG reductase were studied in detail.…”

Section: Discussionmentioning

confidence: 99%

“…In view of the high activity of these enzymes (120 U/g liver [16]), the concentration of CDNB is assumed to be low in situ. Since nitrated aromatic compounds are known as strong inhibitors of glutathione reductase [17] we tested the effect of CDNB on this enzyme in vitro and found a Ki value of 22 pM.…”

Section: Inhibition Of' Glutathione Reductase By I-chloro-24-dinitromentioning

confidence: 99%

Interaction of a glutathione S‐conjugate with glutathione reductase Kinetic and X‐ray crystallographic studies

Bilzer¹,

Krauth‐Siegel²,

Schirmer³

et al. 1984

European Journal of Biochemistry

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1. S-Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73-76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2).2. The interaction of GSSG reductase with a well-studied conjugate, namely S-(2,4-dinitrophenyl)-glutathione and its electrophilic precursor l-chloro-2,4-dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 pM and 22 pM respectively. After prolonged incubation, 1 -chloro-2,4-dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ.3. As shown by X-ray crystallography the major binding site of S-(2,4-dinitrophenyl)-glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.Glutathione disulfide (GSSG) and glutathione S-conjugates show mutual inhibition of transport across the canalicular membrane in perfused rat liver [I] and across the plasma membrane of red blood cells [2]. As reported here, the intrahepatic accumulation of GSSG in the presence of a conjugate is not only a consequence of a lower rate of excretion but in addition caused by an inhibition of GSSG reductase. This flavoprotein [3] is known in atomic detail with regard to sequence [4], three-dimensional structure [5] and stereochemistry of catalysis [6]. The paper demonstrates the inhibition of purified human GSSG reductase by a glutathione thioether, S-(2,4-dinitrophenyl)-glutathione (DNPG), and shows its binding to the enzyme as detected by X-ray analysis. MATERIALS AND METHODS Liver perfusionNon-recirculating hemoglobin-free perfusion of livers from male Wistar rats (180-220 g body weight) fed on stock diet (Altromin, Lage) was performed as in [I]. Experiments were terminated by freeze-clamping the liver tissue for determination of intracellular GSSG [I]. Preparation and assay of S-(2,4-dinitrophenyl) -glutathioneS-(2,4-Dinitrophenyl)-glutathione was synthesized from GSH and l-chloro-2,4-dinitrobenzene (CDNB) using glutathione S-transferases. The thioether was purified by preparative thin-layer chromatography as described by Awasthi et al.

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Glutathione S‐Conjugate Formation from 1‐Chloro‐2,4‐dinitrobenzene and Biliary S‐Conjugate Excretion in the Perfused Rat Liver

Cited by 100 publications

References 17 publications

Kinetic analysis of glutathione in anchored cells with monochlorobimane

Kinetic analysis of glutathione in anchored cells with monochlorobimane

Glutathione S‐conjugates stimulate ATP hydrolysis in the plasma membrane fraction of rat hepatocytes

Interaction of a glutathione S‐conjugate with glutathione reductase Kinetic and X‐ray crystallographic studies

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