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Published on: July 18, 2011
Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells
Janine Mauzeroll1, Allen J Bard
1Department of Chemistry and Biochemistry, University of Texas, 1 University Station, A5300, Austin, TX 78712, USA.
Abstract:
The uptake of menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy. The progression of the in vitro reaction between menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV-visible) behavior. By observing the scanning electrochemical microscope tip current of yeast cells suspended in a menadione-containing solution, the export of the conjugate from the cells with time could be measured. Similar experiments were performed on immobilized yeast cell aggregates stressed by a menadione solution. From the export of the menadione-glutathione conjugate detected at a 1-microm-diameter electrode situated 10 microm from the cells, a flux of about 30,000 thiodione molecules per second per cell was extracted. Numerical simulations based on an explicit finite difference method further revealed that the observation of a constant efflux of thiodione from the cells suggested the rate was limited by the uptake of menadione and that the efflux through the glutathione-conjugate pump was at least an order of magnitude faster.
Insights
Yeast cells expel toxic menadione (2-methyl-1,4-naphthoquinone) as a glutathione complex. Scanning electrochemical microscopy revealed efflux rates limited by menadione uptake, with the export pump operating much faster.
Area of Science:
- Biochemistry
- Cell Biology
- Electrochemistry
Background:
- Menadione (2-methyl-1,4-naphthoquinone) is toxic to yeast cells.
- Glutathione conjugation is a key detoxification pathway in many organisms.
Purpose of the Study:
- To investigate the uptake and expulsion of menadione by yeast cells.
- To quantify the rate of menadione-glutathione conjugate efflux.
- To understand the kinetics of menadione detoxification in yeast.
Main Methods:
- Scanning electrochemical microscopy (SECM) to monitor menadione uptake and efflux.
- Cyclic voltammetry to study the menadione-glutathione reaction kinetics.
- UV-visible spectroscopy for reaction monitoring.
- Numerical simulations using finite difference methods.
Main Results:
- SECM successfully measured the export of the menadione-glutathione conjugate from yeast cells.
- A flux of approximately 30,000 thiodione molecules per second per cell was calculated.
- Numerical simulations indicated that menadione uptake, not efflux, limited the overall detoxification rate.
- The glutathione-conjugate pump's efflux rate was at least ten times faster than menadione uptake.
Conclusions:
- Yeast cells actively export menadione as a glutathione conjugate.
- The rate-limiting step in menadione detoxification is menadione uptake into the cell.
- The glutathione-conjugate efflux pump is highly efficient, operating significantly faster than uptake.
