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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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Mean steady state currents from LSVs of cells incubated with solutions of: 20 mM [Fe(CN)6]3− only (A); Mean steady state currents from LSVs of cells incubated with solutions of 20 mM [Fe(CN)6]3− and 100 μM DCIP (B); and Mean steady state currents from LSVs of cells incubated with solutions of 20 mM [Fe(CN)6]3− and 100 μM GC (C) at 425 mV vs Ag/AgCl.Each group was incubated with no inhibitors, dicumarol (100 μM), 6-AN (100 μM), and both inhibitors. Standard incubation conditions were used. Error bars represent ±1SE (n = 9).
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f6: Mean steady state currents from LSVs of cells incubated with solutions of: 20 mM [Fe(CN)6]3− only (A); Mean steady state currents from LSVs of cells incubated with solutions of 20 mM [Fe(CN)6]3− and 100 μM DCIP (B); and Mean steady state currents from LSVs of cells incubated with solutions of 20 mM [Fe(CN)6]3− and 100 μM GC (C) at 425 mV vs Ag/AgCl.Each group was incubated with no inhibitors, dicumarol (100 μM), 6-AN (100 μM), and both inhibitors. Standard incubation conditions were used. Error bars represent ±1SE (n = 9).

Mentions: The metabolism inhibitors, dicumarol and 6-AN, were used to provide further evidence on the interaction of the mediators with redox sites. Dicumarol affects respiration in a number of ways both increasing and decreasing enzymes involved in these pathways31. Selection of 6-AN was based on its inhibition of 6-phosphogluconate dehydrogenase in the Pentose Phosphate Pathway (PPP), which results in a decrease in NADPH production32. The secondary mediators DCIP and gallocyanine were selected for this study because their interactions with the cell were expected to be straightforward. DCIP is a mediator that has been reported to interact with a limited number of internal redox sites e.g. it has been reported to preferentially interact with the fermentative pathway33 and interacts with respiratory processes only in the presence of inhibitors34. Gallocyanine is an example of a mediator that gives a small response in the double mediator system and has not been reported to interact intracellularly. On the other hand, MD and 2,3,5,6,-TMPD are known to interact with a wide range of cell redox molecules including those in the mitochondria and hence would be expected to give data that was difficult to interpret35. Controls containing [Fe(CN)6]3− and the metabolism inhibitors were also performed. Measurement of the conversion of [Fe(CN)6]3− to [Fe(CN)6]4− for each mediator/inhibitor combination is shown in Figure 6A ([Fe(CN)6]3− only), 6B ([Fe(CN)6]3− and DCIP) and 6C ([Fe(CN)6]3− and gallocyanine). The patterns of responses of each system to the inhibitors were compared to determine the sites of interactions and two-way ANOVAs and a post-hoc Tukey test were used to verify differences or similarities of the signals within and between each group.


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

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

Mean steady state currents from LSVs of cells incubated with solutions of: 20 mM [Fe(CN)6]3− only (A); Mean steady state currents from LSVs of cells incubated with solutions of 20 mM [Fe(CN)6]3− and 100 μM DCIP (B); and Mean steady state currents from LSVs of cells incubated with solutions of 20 mM [Fe(CN)6]3− and 100 μM GC (C) at 425 mV vs Ag/AgCl.Each group was incubated with no inhibitors, dicumarol (100 μM), 6-AN (100 μM), and both inhibitors. Standard incubation conditions were used. Error bars represent ±1SE (n = 9).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Mean steady state currents from LSVs of cells incubated with solutions of: 20 mM [Fe(CN)6]3− only (A); Mean steady state currents from LSVs of cells incubated with solutions of 20 mM [Fe(CN)6]3− and 100 μM DCIP (B); and Mean steady state currents from LSVs of cells incubated with solutions of 20 mM [Fe(CN)6]3− and 100 μM GC (C) at 425 mV vs Ag/AgCl.Each group was incubated with no inhibitors, dicumarol (100 μM), 6-AN (100 μM), and both inhibitors. Standard incubation conditions were used. Error bars represent ±1SE (n = 9).
Mentions: The metabolism inhibitors, dicumarol and 6-AN, were used to provide further evidence on the interaction of the mediators with redox sites. Dicumarol affects respiration in a number of ways both increasing and decreasing enzymes involved in these pathways31. Selection of 6-AN was based on its inhibition of 6-phosphogluconate dehydrogenase in the Pentose Phosphate Pathway (PPP), which results in a decrease in NADPH production32. The secondary mediators DCIP and gallocyanine were selected for this study because their interactions with the cell were expected to be straightforward. DCIP is a mediator that has been reported to interact with a limited number of internal redox sites e.g. it has been reported to preferentially interact with the fermentative pathway33 and interacts with respiratory processes only in the presence of inhibitors34. Gallocyanine is an example of a mediator that gives a small response in the double mediator system and has not been reported to interact intracellularly. On the other hand, MD and 2,3,5,6,-TMPD are known to interact with a wide range of cell redox molecules including those in the mitochondria and hence would be expected to give data that was difficult to interpret35. Controls containing [Fe(CN)6]3− and the metabolism inhibitors were also performed. Measurement of the conversion of [Fe(CN)6]3− to [Fe(CN)6]4− for each mediator/inhibitor combination is shown in Figure 6A ([Fe(CN)6]3− only), 6B ([Fe(CN)6]3− and DCIP) and 6C ([Fe(CN)6]3− and gallocyanine). The patterns of responses of each system to the inhibitors were compared to determine the sites of interactions and two-way ANOVAs and a post-hoc Tukey test were used to verify differences or similarities of the signals within and between each group.

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