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Mediator-assisted simultaneous probing of cytosolic and mitochondrial redox activity in living cells.

Heiskanen, Arto LU ; Spegel, Christer LU ; Kostesha, Natalie LU ; Lindahl, Sofia; Ruzgas, Tautgirdas and Emnéus, Jenny (2009) In Analytical Biochemistry 384(1). p.11-19
Abstract
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 pentose phosphate pathway, respectively.... (More)
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 pentose 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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Analytical Biochemistry
volume
384
issue
1
pages
11 - 19
publisher
Elsevier
external identifiers
  • wos:000263124000002
  • pmid:18812160
  • scopus:56249112260
ISSN
1096-0309
DOI
10.1016/j.ab.2008.08.030
language
English
LU publication?
yes
id
cf72d395-4912-4079-b33a-636927d228e4 (old id 1242702)
date added to LUP
2009-03-09 12:12:27
date last changed
2017-11-12 03:27:13
@article{cf72d395-4912-4079-b33a-636927d228e4,
  abstract     = {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 pentose 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.},
  author       = {Heiskanen, Arto and Spegel, Christer and Kostesha, Natalie and Lindahl, Sofia and Ruzgas, Tautgirdas and Emnéus, Jenny},
  issn         = {1096-0309},
  language     = {eng},
  number       = {1},
  pages        = {11--19},
  publisher    = {Elsevier},
  series       = {Analytical Biochemistry},
  title        = {Mediator-assisted simultaneous probing of cytosolic and mitochondrial redox activity in living cells.},
  url          = {http://dx.doi.org/10.1016/j.ab.2008.08.030},
  volume       = {384},
  year         = {2009},
}