Glyceraldehyde phosphate dehydrogenase oxidation during Cardiac ischemia and reperfusion

Objectives: Protein S-glutathiolation is a predicted mechanism by which protein thiol groups are oxidized during the oxidative stress of ischaemia and reperfusion. We measured protein S-thiolation during ischaemia and reperfusion and investigated the effect of this oxidative modification on the function of GAPDH. Methods: Glutathione was biotinylated (biotin-GSH) and used to probe for protein S-glutathiolation in isolated rat hearts using non-reducing Western blots and streptavidin-HRP. Streptavidin-agarose was used to purify S-glutathiolated proteins and these were identified using N-terminal sequencing and database searching.Results: Little protein S-glutathiolation occurred in control preparations, but this increased 15-fold during reperfusion. Protein S-glutathiolation was attenuated by the antioxidant mercaptopropionylglycine and was shown to occur only during the first minutes of reperfusion. Affinity purification of the S-glutathiolated proteins showed 20 dominant S-glutathiolation substrates. A dominant S-thiolated protein was N-terminally sequenced (VKVGVNGFG) and HPLC peptide mapping gave additional sequence nearer the site of oxidation (TGVFTTMEKA). The first sequence was the N-terminus of GAPDH, and the second a peptide from the same protein starting at residue 96. GAPDH was immunopurified from aerobic, ischemic or reperfused hearts. Maleimidofluorescein labeling of purified GAPDH provided an index of its reduced thiol status. In the absence of DTT, ischemia induced a reduction in the number of free thiols on GAPDH that was reversed on reperfusion. When treated with DTT, the free thiol status of GAPDH could be increased in ischemic but not reperfused samples. Ischemia induced a reduction in GAPDH activity that was partially restored by reperfusion. DTT-treatment reactivated ischemic GAPDH, but had little effect on the activity from reperfused tissue. Mass spectra acquired from aerobic GAPDH preparations were relatively simple whereas spectra from ischemic or reperfused preparations were highly complex, possibly indicative of oxidation by multiple oxidants.Conclusions: Many proteins, including GAPDH, are targets for S-glutathiolation during cardiac oxidative stress. GAPDH oxidation is associated with a loss in reduced cysteine status that correlates with the inactivation of this enzyme