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Free Content Quantitative in vivo measurement of glutathione in Arabidopsis cells

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Summary

A new, non-destructive assay is described to quantify cytoplasmic glutathione (GSH) levels in vivo in single cells or populations of cells from Arabidopsis suspension cultures. Cytoplasmic GSH was labelled with monochlorobimane (MCB) in situ to give a fluorescent GSH–bimane (GSB) conjugate. At low (10–100 µm) concentrations of MCB, labelling was mediated by a glutathione S-transferase, which confers specificity for GSH. HPLC analysis of MCB-labelled low molecular-weight thiols showed that the assay measures the total GSH pool, including the oxidized glutathione. The progress curve for the labelling could be described using Michaelis–Menten kinetics with an apparent KM of 40 µmand Vmax of 470 µmol lcyt−1 min−1. There was no evidence for de novo synthesis of GSH during the labelling period of 2 h, suggesting that control of GSH synthesis is not mediated by feedback control of γ-glutamylcysteine synthetase in this system. The total cellular level of GSH was calculated from the plateau value of the progress curve, after appropriate calibration, as 830–942 nmol g−1 FW. The volume fraction of cytoplasm was measured from serial optical sections of bimane-labelled cells collected by confocal laser scanning microscopy (CLSM) with excitation 442 nm, or two-photon laser scanning microscopy (TPLSM) with excitation 770 nm. A value of 42 ± 3% cytoplasm was determined by manual segmentation, and a value of 37 ± 2% using stereological techniques. Using these figures, values for cytoplasmic [GSH] were estimated to be between 2.7 ± 0.3 and 3.2 ± 0.3 mmfor cell populations. In addition, measurement of GSH levels in individual cells using CLSM and TPLSM gave values of 3.0 ± 0.5 and 3.5 ± 0.7 mm, respectively.
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Keywords: Arabidopsis; fluorimetry; glutathione; glutathione S-transferase; laser scanning microscopy; monochlorobimane; stereology

Document Type: Research Article

Affiliations: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK

Publication date: July 1, 2001

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