Sepharose-insolubilization of the dihydrolipoyl transacetylase core component of the pyruvate dehydrogenase complex: preparation and characterization.
The dihydrolipoyl transacetylase core components of the bovine kidney and heart pyruvate dehydrogenase complexes were covalently attached through the lipoyl moiety to Sepharose by the thiol-crosslinking reagent, N,N'-p-phenylenedimaleimide. In one approach, the N,N-p-phenylenedimaleimide was allowed to react with glutathione which was in turn linked by its N-terminal to Sepharose CL-6B. In addition, we found that N,N-p-phenylenedimaleimide would react directly with Sepharose CL-6B (at undetermined sites) and could be used as the sole bridge in forming a stable linkage of the transacetylase core to Sepharose. With the latter approach the extent of multiple-linkage of the 60-subunit core could more easily be controlled. This should be a generally useful approach for linking proteins with reactive surface thiol residues. Insolubilization of the core of the pyruvate dehydrogenase complex by these methods did not appear to significantly alter the binding of other protein components of the complex, but the catalytic activities of the complex requiring the lipoyl moiety were appreciably altered. Procedures for coupling the transacetylase core to various derivatives of phenylenedimaleimide-Sepharose and techniques described for studying the protein products should be useful in preparation of specialized matrices for both protein purification and the study of protein-protein interactions.
D000123:Acetyltransferases / Q000032:analysis*; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004800:Enzymes, Immobilized / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011768:Pyruvate Dehydrogenase Complex / Q000032:analysis*; D012685:Sepharose; D012995:Solubility
D004800:Enzymes, Immobilized; D011768:Pyruvate Dehydrogenase Complex; D012685:Sepharose; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase