TitleAssessment of endoplasmic reticulum glutathione redox status is confounded by extensive ex vivo oxidation.
Publication TypeJournal Article
Year of Publication2008
AuthorsDixon BM, Heath S-HD, Kim R, Suh JH, Hagen TM
JournalAntioxid Redox Signal
Date Published2008 May
KeywordsAnimals, Endoplasmic Reticulum, Enzyme Inhibitors, Glutathione, Glutathione Disulfide, Iodoacetic Acid, Microsomes, Liver, Oxidation-Reduction, Rats, Rats, Inbred F344

Glutathione (GSH) and glutathione disulfide (GSSG) form the principal thiol redox couple in the endoplasmic reticulum (ER); however, few studies have attempted to quantify GSH redox status in this organelle. To address this gap, GSH and GSSG levels and the extent of protein glutathionylation were analyzed in rat liver microsomes. Because of the likelihood of artifactual GSH oxidation during the lengthy microsomal isolation procedure, iodoacetic acid (IAA) was used to preserve the physiological thiol redox state. Non-IAA-treated microsomes exhibited a GSH:GSSG ratio between 0.7:1 to 1.2:1 compared to IAA-treated microsomes that yielded a GSH:GSSG redox ratio between 4.7:1 and 5.5:1. The majority of artifactual oxidation occurred within the first 2 h of isolation. Thus, the ER GSH redox ratio is subject to extensive ex vivo oxidation and when controlled, the microsomal GSH redox state is significantly higher than previously believed. Moreover, in vitro studies showed that PDI reductase activity was markedly increased at this higher thiol redox ratio versus previously reported GSH:GSSG ratios for the ER. Lastly, we show by both HPLC and Western blot analysis that ER proteins are highly resistant to glutathionylation. Together, these results may necessitate a re-evaluation of GSH and its role in ER function.

Alternate JournalAntioxid. Redox Signal.
PubMed ID18205546
PubMed Central IDPMC3220945
Grant ListP01 AT 002034-01 / AT / NCCIH NIH HHS / United States
R01 AG017141 / AG / NIA NIH HHS / United States
R01 2 AG 17141 / AG / NIA NIH HHS / United States
ES 00210 / ES / NIEHS NIH HHS / United States
R01 AG017141-01A1 / AG / NIA NIH HHS / United States
P01 AT002034 / AT / NCCIH NIH HHS / United States
P30 ES000210 / ES / NIEHS NIH HHS / United States