Mediator-assisted Simultaneous probing of Cytosolic and Mitochondrial Redox activity in living cells

Arto Heiskanen, Christer Spegel, Natalie Kostesha, S. Lindahl, T. Ruzas, Jenny Emnéus

    Research output: Contribution to journalJournal articleResearchpeer-review


    This work describes an electron transfer mediator-assisted amperometric flow injection method for assessing redox enzyme activity in different subcellular compartments of the phosphoglucose isomerase deletion mutant strain of Saccharomyces cerevisiae, EBY44. The method is demonstrated using the ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pen-rose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric in vitro assays show no difference between the cofactors. Respiratory competent cells show cytosolic inhibition only when NADPH is produced, whereas production of NADH reveals uncoupling at low dicoumarol concentrations and inhibition of complexes III and IV at higher concentrations. Spectrophotometric assays only indicate the presence of cytosolic inhibition regardless of the reduced cofactor used. This article shows the applicability of the amperometric method and emphasizes the significance of determining biological effects of chemicals in living cells.
    Original languageEnglish
    JournalAnalytical Biochemistry
    Issue number1
    Pages (from-to)11-19
    Publication statusPublished - 2009


    • Double mediator system
    • Assessment of redox activity
    • Living cells
    • Saccharomyces cerevisiae


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