Refolding and reconstitution studies on the transacetylase-protein X (E2/X) subcomplex of the mammalian pyruvate dehydrogenase complex: evidence for specific binding of the dihydrolipoamide dehydrogenase component to sites on reassembled E2

Biochemistry
R G McCartneyJ G Lindsay

Abstract

Reconstitution studies have been conducted on the dihydrolipoamide acetyltransferase-protein X core subcomplex of the mammalian pyruvate dehydrogenase complex. GdnHCl-induced dissociation of this core is an ordered cooperative event involving formation of specific lower-Mr intermediates corresponding to dihydrolipoamide acetyltransferase trimers and monomers. Recovery profiles of the dihydrolipoamide acetyltransferase-protein X core, unfolded in 6 M GdnHCl prior to the removal of denaturant by either (a) slow dialysis or (b) rapid dilution, demonstrated rapid initial reappearance of substantial levels of dihydrolipoamide acetyltransferase activity with complete recovery occurring in 4-6 h. Immunological analysis of reconstituted cores revealed reduced levels of protein X (approximately 30-35%) after slow dialysis and the total absence of this component following rapid dilution. The dihydrolipoamide acetyltransferase core, devoid of protein X, was unable to sustain overall complex activity when reconstituted with stoichiometric amounts of its companion pyruvate decarboxylase and dihydrolipoamide deydrogenase components, whereas the protein X-depleted core could sustain 30-35% of control activity. Further reconstitution analyses ...Continue Reading

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Citations

Jan 5, 2002·Journal of Peptide Science : an Official Publication of the European Peptide Society·F BertolaG Precigoux
Sep 5, 2002·Molecular Genetics and Metabolism·Runu DeyCecile Marsac
Jan 7, 2014·Amino Acids·Wei-Qiang ChenGert Lubec
May 21, 2003·Proceedings of the National Academy of Sciences of the United States of America·Yingqi GuJames K Stoops
Apr 7, 2010·Journal of Molecular Biology·S VijayakrishnanO Byron

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