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Quantitative in vivo measurement of glutathione in Arabidopsis cells
Author(s) -
Meyer Andreas J.,
May Mike J.,
Fricker Mark
Publication year - 2001
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2001.01071.x
Subject(s) - glutathione , cytoplasm , in vivo , confocal microscopy , chemistry , labelling , biophysics , conjugate , biochemistry , biology , microbiology and biotechnology , enzyme , genetics , mathematical analysis , mathematics
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 µ m ) 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 K M of 40 µ m and V max of 470 µmol l cyt −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 m m for 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 m m , respectively.