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

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

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.

Detaljer

Författare
  • Arto Heiskanen
  • Christer Spegel
  • Natalie Kostesha
  • Sofia Lindahl
  • Tautgirdas Ruzgas
  • Jenny Emnéus
Enheter & grupper
Forskningsområden

Ämnesklassifikation (UKÄ) – OBLIGATORISK

  • Industriell bioteknik
  • Analytisk kemi
Originalspråkengelska
Sidor (från-till)11-19
TidskriftAnalytical Biochemistry
Volym384
Utgåva nummer1
StatusPublished - 2009
PublikationskategoriForskning
Peer review utfördJa