Monochlorobimane Does Not Selectively Label Glutathione in Peripheral Blood Mononuclear Cells

Ven, A.J.A.M. van der; Mier, P.D.; Peters, W.H.M.; Dolstra, Harry; Erp, P.E.J. van; Koopmans, P.P.; Meer, J.W.M. van der

doi:10.1006/abio.1994.1081

Cited by 34 publications

(17 citation statements)

References 16 publications

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“…Highresolution imaging showed the presence of residual amounts of GSH in rml1 embryos and in the primary root of rml1 seedlings. MCB has been reported to nonspecifically label thiols in situ in the absence of a specific glutathione S-transferase capable of catalyzing the conjugation reaction with GSH (van der Ven et al, 1994). In this study, however, the absence of a detectable MCB-dependent fluorescence signal in homozygous gsh1-T embryos demonstrates that almost all the MCB-dependent fluorescence signal arises from conjugation with GSH, with no other low-M r thiols or protein thiols labeled to a detectable level under the conditions used.…”

Section: Discussionmentioning

confidence: 53%

Maturation of Arabidopsis Seeds Is Dependent on Glutathione Biosynthesis within the Embryo

et al. 2006

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Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, g-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoter::b-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.

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Section: Discussionmentioning

confidence: 53%

Maturation of Arabidopsis Seeds Is Dependent on Glutathione Biosynthesis within the Embryo

et al. 2006

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“…Despite these useful properties, there have been a number of limitations that affect the utility of mBCl for GSH evaluation. For example, GST isozyme heterogeneity in different cells combined with isozyme differences in reactivity toward mBCl can lead to incomplete mBCI-GSH conjugate formation (32). Dissimilarity in the availability of the different pools of GSH [e.g., reduced availability of GSH in the mitochondrial GSH pool (19) that constitutes about 10% of the total GSH pool (25)j in intact cells; compartmentalization or loss of the mBC1-GSH conjugate from the cell (32); and other variables, such as dye concentration, loading time, and temperature, may influence cellular fluorescence obtained with mBCl(23).…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, the compartmentalization and availability of different pools of GSH in intact cells [such as mitochondria ( 18) or nuclei (6)], loss of the mBCI-GSH conjugate from the cell (32), and other variables may influence cellular fluorescence obtained with mBCl (13,23 (12). The COLO-316/S line was used to generate a COLO-316/DDP line, which is resistant to the chemotherapeutic drug cisplatin (DDP), by intermittent incremental exposure to cisplatin (0.1, 1.0, 5, 10 pM).…”

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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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“…Monochlorobimane (MCB), a permeable and uv-excitable thiol adduct fluorescent probe (Ex 380/Em 480), has also been used to quantify GSH in cells; however, this probe may have some limitations, especially in lymphocytes. MCB has low reactivity with thiols, including GSH; therefore, it must be used at high concentrations to achieve binding with most of the GSH and binding is not restricted to GSH (Hedley and Chow, 1994;van der Ven et al, 1994). At lower concentrations, binding is more dependent on glutathione S-transferase, and the concentration and reactivities of the isoenzymes can vary (Ublacker et al, 1991;Cook et al, 1991).…”

Section: Detection Of Oxidant-induced Injury To Humanmentioning

confidence: 99%