Immunology, biosynthesis and in vivo assembly of the branched‐chain 2‐oxoacid dehydrogenase complex from bovine kidney

Abstract

Specific, polyclonal antisera have been raised to the native branched-chain 2-oxoacid dehydrogenase complex (BCOADC) from bovine kidney and each of its three constituent enzymes: E1, the substrate-specific 2-oxoacid dehydrogenase; E2, the multimeric dihydrolipoamide acyltransferase 'core' enzyme and E3, dihydrolipoamide dehydrogenase. Purified BCOADC, isolated by selective poly(ethyleneglycol) precipitation and hydroxyapatite chromatography, contains only traces of endogenous E3 as detected by a requirement for this enzyme in assaying overall complex activity and by immunoblotting criteria. A weak antibody response was elicited by the E1 beta subunit relative to the E2 and E1 alpha polypeptides employing either purified E1 or BCOADC as antigens. Anti-BCOADC serum showed no cross-reaction with high levels of pig heart E3 indicating the absence of antibody directed against this component. However, immunoprecipitates of mature BCOADC from detergent extracts of NBL-1 (bovine kidney) or PK-15 (porcine kidney) cell lines incubated for 3-4 h in the presence of [35S]methionine contained an additional 55,000-Mr species which was identified as E3 on the basis of immunocompetition studies. Accumulation of newly synthesised [35S]methionine-labelled precursors for E2, E1 alpha and E3 was achieved by incubation of PK-15 cells for 4 h in the presence of uncouplers of oxidative phosphorylation. Pre-E2 exhibited an apparent Mr value of 56,500, pre-E1 alpha, 49,000 and pre-E3, 57,000 compared to subunit Mr values of 50,000, 46,000 and 55,000, respectively, for the mature polypeptides. Thus, like the equivalent lipoate acyltransferases of the mammalian pyruvate dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) complexes, pre-E2 of BCOADC characteristically contains an extended presequence. In NBL-1 cells, pre-E2 was found to be unstable since no cytoplasmic pool of this precursor could be detected; moreover, processed E1 alpha was not assembled into intact BCOADC as evidenced by the absence of E2 or E3 in immunoprecipitates with anti-(BCOADC) serum after a 45-min 'chase' period in the absence of uncoupler. Dihydrolipoamide dehydrogenase (E3), in its precursor state, was not present in immune complexes with anti-(BCOADC) serum, indicating that its co-precipitation with mature complex is by virtue of its high affinity for assembled complex in vivo whereas no equivalent interaction of pre-E3 with its companion precursors occurs prior to mitochondrial import.