Immunological and biosynthetic studies on the mammalian 2-oxoglutarate dehydrogenase multienzyme complex.
High-titre, monospecific, polyclonal antisera have been raised against purified mitochondrial 2-oxoglutarate dehydrogenase complex (OGDC) from ox heart and two of its three constituent enzymes, 2-oxoglutarate dehydrogenase (E1) and lipoyl succinyltransferase (E2). These specific antisera have been employed to monitor molecular events in the biosynthesis, import and maturation of this multimeric assembly. Lipoamide dehydrogenase (E3) elicits a poor antibody response in comparison to the other polypeptides of the complex. In cultured pig kidney cells (PK-15), incubated with [35S]methionine in the presence of uncouplers of oxidative phosphorylation, appearance of stable higher-Mr forms of the individual enzymes can be detected by specific immunoprecipitation and fluorographic analysis. In the case of 2-oxoglutarate dehydrogenase, E1, the initial cytoplasmic translation product has a subunit Mr value of 1500-3000 greater than in the mature enzyme while the precursor of the lipoyl succinyltransferase, E2, contains an additional sequence of Mr 6000-8000. Competition studies have revealed the immunological similarity of the precursor molecules to the native subunits. On removal of uncouplers, processing of accumulated precursors is rapidly initiated and is complete within 40 min. Interestingly, antiserum to native 2-oxoglutarate dehydrogenase complex fails to recognise E2 precursor molecules (pre-E2), which can be immunoprecipitated, however, by antibodies raised against the denatured E2 subunit. It is concluded that pre-E2 is conformationally dissimilar to native E2, which exists normally as a highly ordered, multimolecular aggregate in the native complex.
D000818:Animals; D002417:Cattle; D002460:Cell Line; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000378:metabolism; D007120:Immunochemistry; D007655:Ketoglutarate Dehydrogenase Complex / Q000096:biosynthesis*; D007658:Ketone Oxidoreductases / Q000096:biosynthesis*; D007668:Kidney / Q000378:metabolism; D008297:Male; D008929:Mitochondria, Heart / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000096:biosynthesis*; D011487:Protein Conformation; D051381:Rats; D011915:Rats, Inbred BUF; D013552:Swine
D004792:Enzyme Precursors; D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex