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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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Diagrammatic representation of the possible mechanisms by which a hydrophilic reporter mediator ([Fe(CN)6]3−) and a secondary mediator can interact with eukaryote cells and with each other. = tPMET proteins.
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f1: Diagrammatic representation of the possible mechanisms by which a hydrophilic reporter mediator ([Fe(CN)6]3−) and a secondary mediator can interact with eukaryote cells and with each other. = tPMET proteins.

Mentions: Electrochemical mediators can accept electrons from a eukaryote cell via a number of pathways. A hydrophilic mediator cannot cross the cell membrane and is restricted to interacting with tPMETs from the periplasm (Figure 1, pathway 1). An example is ferricyanide ([Fe(CN)6]3−), which is reduced to ferrocyanide ([Fe(CN)6]4−) by a ferri-reductase tPMET system that is embedded in the plasma membrane of Saccharomyces cerevisiae1 and Merker et al. have shown that the reduction of toluidine blue O polyacrylamide (TBOP), a hydrophilic mediator, parallels the oxidation of cytoplasmic NADH2. A double mediator system (Figure 1, pathway 2) comprising lipophilic and hydrophilic mediators enables intracellular redox systems to be accessed. Lipophilic mediators can cross the cell membrane and interact with intracellular redox centres, become reduced and diffuse or are transported out of the cell to transfer electrons to a hydrophilic reporter mediator. In practice, lipophilic mediators usually cannot be used as the sole mediator because low aqueous solubility limits the mediator concentration and hence the magnitude of the signal. Additionally, often their limited stability in one redox state limits the techniques that can be used to probe the system. Double mediator systems using lipophilic menadione (MD) or 2,3,5,6-tetramethylphenylenediamine (2,3,5,6-TMPD), in conjunction with [Fe(CN)6]3− have been used to investigate Chinese hamster ovary cells (CHO)3 and S.cerevisiae345. The large signals observed in these studies could only have originated from intracellular redox molecules including those in the mitochondria. There are two other mechanisms by which a lipophilic mediator could transfer electrons to a reporter mediator. In the first, the reduced lipophilic mediator interacts with intracellular tPMET sites, which then passes the electron across the membrane to reduce the reporter mediator (Figure 1, pathway 3). This will only occur if the potential of the mediator is lower than the internal site of the tPMET. In the second mechanism (Figure 1, pathway 4), the mediator is reduced extracellularly by a tPMET system that then transfers its electrons to the reporter mediator6. Note: the signal size from pathway 4 will be very small relative to signals from pathway 2 because pathway 4 is only accessing tPMETs whereas in pathway 2, the mediator accesses intracellular redox molecules. It is likely that more than one of these pathways is operating simultaneously in any double mediator system investigation.


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

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

Diagrammatic representation of the possible mechanisms by which a hydrophilic reporter mediator ([Fe(CN)6]3−) and a secondary mediator can interact with eukaryote cells and with each other. = tPMET proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Diagrammatic representation of the possible mechanisms by which a hydrophilic reporter mediator ([Fe(CN)6]3−) and a secondary mediator can interact with eukaryote cells and with each other. = tPMET proteins.
Mentions: Electrochemical mediators can accept electrons from a eukaryote cell via a number of pathways. A hydrophilic mediator cannot cross the cell membrane and is restricted to interacting with tPMETs from the periplasm (Figure 1, pathway 1). An example is ferricyanide ([Fe(CN)6]3−), which is reduced to ferrocyanide ([Fe(CN)6]4−) by a ferri-reductase tPMET system that is embedded in the plasma membrane of Saccharomyces cerevisiae1 and Merker et al. have shown that the reduction of toluidine blue O polyacrylamide (TBOP), a hydrophilic mediator, parallels the oxidation of cytoplasmic NADH2. A double mediator system (Figure 1, pathway 2) comprising lipophilic and hydrophilic mediators enables intracellular redox systems to be accessed. Lipophilic mediators can cross the cell membrane and interact with intracellular redox centres, become reduced and diffuse or are transported out of the cell to transfer electrons to a hydrophilic reporter mediator. In practice, lipophilic mediators usually cannot be used as the sole mediator because low aqueous solubility limits the mediator concentration and hence the magnitude of the signal. Additionally, often their limited stability in one redox state limits the techniques that can be used to probe the system. Double mediator systems using lipophilic menadione (MD) or 2,3,5,6-tetramethylphenylenediamine (2,3,5,6-TMPD), in conjunction with [Fe(CN)6]3− have been used to investigate Chinese hamster ovary cells (CHO)3 and S.cerevisiae345. The large signals observed in these studies could only have originated from intracellular redox molecules including those in the mitochondria. There are two other mechanisms by which a lipophilic mediator could transfer electrons to a reporter mediator. In the first, the reduced lipophilic mediator interacts with intracellular tPMET sites, which then passes the electron across the membrane to reduce the reporter mediator (Figure 1, pathway 3). This will only occur if the potential of the mediator is lower than the internal site of the tPMET. In the second mechanism (Figure 1, pathway 4), the mediator is reduced extracellularly by a tPMET system that then transfers its electrons to the reporter mediator6. Note: the signal size from pathway 4 will be very small relative to signals from pathway 2 because pathway 4 is only accessing tPMETs whereas in pathway 2, the mediator accesses intracellular redox molecules. It is likely that more than one of these pathways is operating simultaneously in any double mediator system investigation.

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