Quaternary structure of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus studied by a new reversible cross-linking procedure with bis(imidoesters).
The bis(amidine) cross-links formed between protein subunits by treating them with bis(imidoesters) were found to be rapidly broken by exposing the cross-linked proteins to methylamine buffers containing the aprotic solvent acetonitrile. This cleavage step could be introduced between the two dimensions of a diagonal gel electrophoretic separation of cross-linked proteins to facilitate identification of the contributors to a cross-linked species. Tests with the tetrameric enzyme glyceraldehyde-3-phosphate dehydrogenase demonstrated the simplicity and effectiveness of the technique. When the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was treated with a range of bis(imidoesters), from dimethyl succinimidate to dimethyl suberimidate, the most informative set of products was obtained with dimethyl glutarimidate. The longer bis(imidoesters) caused too extensive cross-linking of the enzyme subunits, although the beta chain of the pyruvate decarboxylase component always appeared to be the most resistant. Almost all the cross-linked species up to pentamers of the lipoate acetyltransferase polypeptide chain (apparent Mr approximately 280 000) were identified by means of the diagonal gel electrophoretic procedure after cleavage of the cross-links. The introduction of the methylamine cleavage step enables the bis(imidoester) for such experiments to be selected purely for the efficacity of its cross-linking reaction with the protein and dispenses with the need to incorporate a specially cleavable bond in the reagent.
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D007096:Imidoesters; D046911:Macromolecular Substances; D008970:Molecular Weight; D011768:Pyruvate Dehydrogenase Complex
D007096:Imidoesters; D046911:Macromolecular Substances; D011768:Pyruvate Dehydrogenase Complex; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases