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. Author manuscript; available in PMC: 2012 Jan 1.
Published in final edited form as: Free Radic Biol Med. 2010 Oct 21;50(1):14–26. doi: 10.1016/j.freeradbiomed.2010.10.694

Figure 5. 4-HNE-mediated adduction of the individual GCL subunits prevents heterodimerization and GCL holoenzyme formation.

Figure 5

Purified recombinant GCLC and GCLM were incubated separately with (A and B) the indicated molar ratio of 4-HNE or (C and D) 100X molar excess of 4-HNE for 30 min at 37°C prior to mixing at a 1:1 molar ratio to form GCL holoenzyme. (A and C) GCL holoenzyme formation was assessed by native PAGE and immunoblotting for GCLC (top panel). The relative levels of the individual subunits were assessed by SDS-PAGE and immunoblotting for GCLC or GCLM (bottom panels). (B and D) GCL activity was determined by the fluorescence-based NDA assay (* p<0.001, ns = not significant).