Assessment of in situ cellular glutathione labeling with naphthalene-2,3-dicarboxaldehyde using high-performance liquid chromatography.

We have presently studied a dialdehydic reagent, i.e. naphthalene-2,3-dicarboxaldehyde (NDA), as a fluorogenic probe for the labeling of intracellular reduced glutathione (GSH), using a yeast strain Candida albicans as a cell model. Chemical reactivity of NDA with both amino and sulfhydryl groups of the GSH molecule leads to a highly selective detection. Moreover, fluorescence properties of the resulting adduct fit well with most of modern instruments adapted for in situ measurements, and equipped with an argon laser. After incubation of cells with 100 microM of NDA for 20 min, cells were harvested and corresponding lysates obtained after a freezing cycle, were suspended in 0.2M borate buffer pH 9.2 and analysed with HPLC (column: Spherisorb ODS-2 (125 mm x 4.6 mm i.d.) 5 microm; mobile phase: methanol-0.01 M phosphate buffer pH 6.5 (20:80, v/v) at a flow rate of 0.8 mL min(-1); spectrofluorimetric detection: lambda(exc)=430 nm and lambda(em)=530 nm). The GSH-NDA adduct was identified in the yeast strain extracts using the reported HPLC technique and quantified versus a calibration curve of NDA derivatized with an excess of GSH (linearity range: 9-230 nM). The cell loading step of the free probe NDA and the extraction efficiency of the resulting NDA-GSH adduct were optimized.

Cadmium-responsive thiols in the ectomycorrhizal fungus Paxillus involutus.

Molecular and cellular mechanisms underlying the sustained metal tolerance of ectomycorrhizal fungi are largely unknown. Some of the main mechanisms involved in metal detoxification appear to involve the chelation of metal ions in the cytosol with thiol-containing compounds, such as glutathione, phytochelatins, or metallothioneins. We used an improved high-performance liquid chromatography method for the simultaneous measurement of thiol-containing compounds from cysteine and its derivatives (gamma-glutamylcysteine, glutathione) to higher-molecular-mass compounds (phytochelatins). We found that glutathione and gamma-glutamylcysteine contents increased when the ectomycorrhizal fungus Paxillus involutus was exposed to cadmium. An additional compound with a 3-kDa molecular mass, most probably related to a metallothionein, increased drastically in mycelia exposed to cadmium. The relative lack of phytochelatins and the presence of a putative metallothionein suggest that ectomycorrhizal fungi may use a different means to tolerate heavy metals, such as Cd, than do their plant hosts.