Characterising structural and functional differences between catalytically active and de-active forms of mammalian mitochondrial complex I

The cross-linking reagents; EEDQ (N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline), EMCS (N-(e-maleimidocaproyloxy)succinimide ester, Sulpho-EMCS (N-(e-maleimidocaproyloxy)sulphosuccinimide ester), DSS (disuccinimidyl suberate) and DSP (dithiobis(succinimidyl propionate) were selected for their different chemical specificities and spacer arm lengths varying from zero- to 12 A. Complex I was isolated by BN-PAGE (Blue native polyacrylamide gel electrophoresis) and subunits were separated by dSDS-PAGE (double SDS polyacrylamide gel electrophoresis) for silver staining.<br><br>The same cross-linked products with cytochrome c oxidase (complex IV) were found with crosslinkers EEDQ and EMCS irrespective of whether complex 1 was treated for the predominantly active- or the predominantly de-active form.

Complex IV subunit I, subunit 4 isoform I and subunit 7A-related protein formed cross-linked products with complex I treated with zero and 9.4 A cross-linking reagents, suggesting a close association of complex I and IV. Complex I also formed cross-linked products with complex II (succinate dehydrogenase/succinate ubiquinone oxidoreductase) subunit II, and an unidentified target, the significance of which, remains to be determined.<br><br>Active and de-active form complex I rat brain SMPs were treated with the S- nitrosothiols: GSNO (S-nitrosoglutathione) and SNAP (S-nitroso-N-acetylpenicillamine).

Any sites of S-nitrosation that differed between the two forms of complex I were determined by differences in fluorescent intensity following a DIGE (difference gel electrophoresis) approach.<br><br>Young and old mouse heart and brain were isolated by a simple membrane isolation technique. Significant differences in complex 1 and complex IV activity were determined with age.