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Electrochemical detection of intracellular and cell membrane redox systems in Saccharomyces cerevisiae.

Rawson FJ, Downard AJ, Baronian KH - Sci Rep (2014)

Bottom Line: After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells.Four of the mediators inhibit electron transfer from S. cerevisiae.Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Biophysics and Surfaces Analysis, School of Pharmacy, University of Nottingham, University Park, Nottingham B15 2TT UK [2] Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.

ABSTRACT
Redox mediators can interact with eukaryote cells at a number of different cell locations. While cell membrane redox centres are easily accessible, the redox centres of catabolism are situated within the cytoplasm and mitochondria and can be difficult to access. We have systematically investigated the interaction of thirteen commonly used lipophilic and hydrophilic mediators with the yeast Saccharomyces cerevisiae. A double mediator system is used in which ferricyanide is the final electron acceptor (the reporter mediator). After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells. The plateau current at 425 mV vs Ag/AgCl gives the analytical signal. The results show that five of the mediators interact with at least three different trans Plasma Membrane Electron Transport systems (tPMETs), and that four mediators cross the plasma membrane to interact with cytoplasmic and mitochondrial redox molecules. Four of the mediators inhibit electron transfer from S. cerevisiae. Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

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Related in: MedlinePlus

Plot of mean steady state currents measured from linear sweep voltammograms at 425 mV vs Ag/AgCl obtained for solutions of 20 mM [Fe(CN)6]3− and 100 μM secondary mediator, using standard incubation and assay conditions.Each current has been corrected with the acellular control values; error bars represent ±1SE (n = 9 except for [Fe(CN)6]3− + cells and [Fe(CN)6]3− controls, n = 33).
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f3: Plot of mean steady state currents measured from linear sweep voltammograms at 425 mV vs Ag/AgCl obtained for solutions of 20 mM [Fe(CN)6]3− and 100 μM secondary mediator, using standard incubation and assay conditions.Each current has been corrected with the acellular control values; error bars represent ±1SE (n = 9 except for [Fe(CN)6]3− + cells and [Fe(CN)6]3− controls, n = 33).

Mentions: The interactions of the thirteen trial or ‘secondary' mediators with cells were quantified by performing double mediator experiments with [Fe(CN)6]3− as the reporter mediator. Cells, trial mediator and reporter mediator were incubated for 1 h as described above. Incubations without cells i.e. acellular controls were also performed for each double mediator combination. At the end of incubation, solution pH was measured, cells, if present, were removed by centrifugation and the supernatants were analysed using steady-state LSV. The voltammograms produced were similar to those shown in Figure 2. The steady state anodic currents were measured at 425 mV, giving the relative amounts of [Fe(CN)6]3− converted to [Fe(CN)6]4− in each experiment. Figure 3 shows the mean steady state anodic currents for the thirteen secondary mediators and the controls. The currents were blank-corrected by subtracting the mean steady state anodic currents of the acellular trials. For the single mediator system i.e. [Fe(CN)6]3− only, the anodic currents for [Fe(CN)6]4− with and without cells are the mean of eleven replicates (33 measurements). An ANOVA shows that there is a significant difference between the with-cells and without-cells trials (p < 0.00001). In the double mediator systems, the amount of [Fe(CN)6]3− converted to [Fe(CN)6]4− varies widely.


Electrochemical detection of intracellular and cell membrane redox systems in Saccharomyces cerevisiae.

Rawson FJ, Downard AJ, Baronian KH - Sci Rep (2014)

Plot of mean steady state currents measured from linear sweep voltammograms at 425 mV vs Ag/AgCl obtained for solutions of 20 mM [Fe(CN)6]3− and 100 μM secondary mediator, using standard incubation and assay conditions.Each current has been corrected with the acellular control values; error bars represent ±1SE (n = 9 except for [Fe(CN)6]3− + cells and [Fe(CN)6]3− controls, n = 33).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048887&req=5

f3: Plot of mean steady state currents measured from linear sweep voltammograms at 425 mV vs Ag/AgCl obtained for solutions of 20 mM [Fe(CN)6]3− and 100 μM secondary mediator, using standard incubation and assay conditions.Each current has been corrected with the acellular control values; error bars represent ±1SE (n = 9 except for [Fe(CN)6]3− + cells and [Fe(CN)6]3− controls, n = 33).
Mentions: The interactions of the thirteen trial or ‘secondary' mediators with cells were quantified by performing double mediator experiments with [Fe(CN)6]3− as the reporter mediator. Cells, trial mediator and reporter mediator were incubated for 1 h as described above. Incubations without cells i.e. acellular controls were also performed for each double mediator combination. At the end of incubation, solution pH was measured, cells, if present, were removed by centrifugation and the supernatants were analysed using steady-state LSV. The voltammograms produced were similar to those shown in Figure 2. The steady state anodic currents were measured at 425 mV, giving the relative amounts of [Fe(CN)6]3− converted to [Fe(CN)6]4− in each experiment. Figure 3 shows the mean steady state anodic currents for the thirteen secondary mediators and the controls. The currents were blank-corrected by subtracting the mean steady state anodic currents of the acellular trials. For the single mediator system i.e. [Fe(CN)6]3− only, the anodic currents for [Fe(CN)6]4− with and without cells are the mean of eleven replicates (33 measurements). An ANOVA shows that there is a significant difference between the with-cells and without-cells trials (p < 0.00001). In the double mediator systems, the amount of [Fe(CN)6]3− converted to [Fe(CN)6]4− varies widely.

Bottom Line: After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells.Four of the mediators inhibit electron transfer from S. cerevisiae.Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Biophysics and Surfaces Analysis, School of Pharmacy, University of Nottingham, University Park, Nottingham B15 2TT UK [2] Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.

ABSTRACT
Redox mediators can interact with eukaryote cells at a number of different cell locations. While cell membrane redox centres are easily accessible, the redox centres of catabolism are situated within the cytoplasm and mitochondria and can be difficult to access. We have systematically investigated the interaction of thirteen commonly used lipophilic and hydrophilic mediators with the yeast Saccharomyces cerevisiae. A double mediator system is used in which ferricyanide is the final electron acceptor (the reporter mediator). After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells. The plateau current at 425 mV vs Ag/AgCl gives the analytical signal. The results show that five of the mediators interact with at least three different trans Plasma Membrane Electron Transport systems (tPMETs), and that four mediators cross the plasma membrane to interact with cytoplasmic and mitochondrial redox molecules. Four of the mediators inhibit electron transfer from S. cerevisiae. Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

Show MeSH
Related in: MedlinePlus