Component X, the recently recognised subunit of mammalian pyruvate dehydrogenase complex, was shown by immune blotting to be present in all of nine tissues dissected from rat. This finding indicated that component X was not an isoenzyme of the lipoate acetyltransferase (E2) associated with one or a limited number of tissues.Native pyruvate dehydrogenase complex was shown to bind IgG raised to isolated component X, indicating that there were at least some regions of the X subunit exposed at the periphery of the complex.Lipoyl groups of ox heart pyruvate dehydrogenase complex were specifically cross-linked by reaction with phenylene-o-bismaleimide in the presence of pyruvate and the subunits contributing to the products of crosslinking were identified by immune blotting. Species with very high MI containing both E2 and component X, were formed in high yield, as well as apparent E2/E2 and E2/X dimers and trimers and an X/X dimer. These results showed that acetylated lipoyl groups of different E2 and X subunits were able to interact in all possible combinations.The types of cross-linked E2 products formed suggested that two thiols, reactible with phenylene-o-bismaleimide, were rapidly generated in the presence of pyruvate. The results were most easily explained by the presence of two acetylatable lipoyl groups on each E2 polypeptide.Pyruvate dehydrogenase complex (PDC) is responsible for the conversion of pyruvate to acetyl-CoA with the overall reactionIn mammalian systems the enzyme is located in the mitochondrion and is a large multienzyme complex of approximate M I = 8.5 x lo6 formed by an assembly of 60 lipoate acetyltransferase subunits (E2) arranged as a pentagonal dodecahedron, bound to which are multiple copies of pyruvate dehydrogenase (El ) and lipoamide dehydrogenase (E3) subunits [l]. Also bound to the pyruvate dehydrogenase complex, although in lower proportions, is a specific kinase involved in regulation of the complex activity by means of covalent modification. The complex is inactivated by the intrinsic kinase, which phosphorylates the Ela subunit and reactivation is achieved by the action of pyruvate dehydrogenase phosphatase [2]. The phosphatase activity does not copurify with the complex. An additional subunit of MI 50000 has recently been identified and has been designated protein or component X [3,4]. Similarly to E2, component X contains covalently bound lipoic acid [5] which is acetylated in the presence of pyruvate or acetyl-CoA and may be reduced by NADH [6]. Immunological characterisation and comparison of peptides produced by limited proteolysis has indicated that component X is unlikely to be derived from E2 by proteolytic cleavage of the E2 polypeptide [3, 61.Studies employing electron microscopy have shown that the lipoyl groups on E2 subunits of PDC are located on peripherally extended regions of polypeptide [7] and analysis with proton NMR show these to be highly mobile relative to other parts of the complex [S]. This mobility would allow interaction of the lipoic acid at spatially...

An immunological analysis has been conducted of early events in the biosynthesis, import and assembly of the mammalian pyruvate dehydrogenase complex (PDC). For this purpose, monospecific polyclonal antisera were produced against the intact assembly from ox heart, Mr 8.5 x 10(6), and each of its component polypeptides, E1 alpha, E1 beta, E2, E3 and protein X. Optimal detergent-based incubation mixtures were developed for obtaining clean immunoprecipitation of PDC polypeptides and their precursors from [35S]methionine-labelled extracts of PK-15 (pig kidney), NBL-1 (bovine kidney) and BRL (Buffalo Rat liver) cells. In PK-15 cells, independent higher Mr species, corresponding to precursors of the E2, E1 alpha and E1 beta subunits of PDC, could be detected by immune precipitation and fluorography after incubation of intact cells for 4 h with [35S]methionine and 1-2 mM-2,4-dinitrophenol or 10-15 microM-carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Similar precursor states could be observed in uncoupler-treated BRL or NBL-1 cells. Pre-E1 alpha, pre-E1 beta and also pre-E3, have signal sequences in the Mr range 1500-3000 while pre-E2 contains a long additional segment of Mr 7000-9000. All of these forms exhibit similar kinetics of processing to the mature subunits with a transit time of 10-12 min. In NBL-1 cells, E3 is present in the immune complexes formed with anti-PDC serum whereas this is not the case in PK-15 cells. Thus, there are significant variations in the affinity of lipoamide dehydrogenase (E3) for the E2 core structure in different species. Pre-E1 alpha accumulates only poorly in PK-15 cells and is aberrantly processed on removal of uncoupler. This precursor is markedly more stable in NBL-1 and BRL cells. The lack of detection of a precursor form of component X is also discussed.

The mammalian pyruvate dehydrogenase complex, M , 8.5 x lo", contains an additional tightly bound 50 000-M , polypeptide, Component X, which copurifies with the intact assembly.Small amounts of the individual E2 and X polypeptides were obtained by elution of the protein bands from SDS/polyacrylamide gels. One-dimensional peptide mapping studies with ' 251-labelled lipoyl acetyltransferase (E2) and component X subunits indicate that these two proteins are structurally distinct entities. Similar analysis of purified subunits, initially radiolabelled in the intact complex in the presence of [2-14C]pyruvate and N-ethyl-[2,3-*4C]maleimide confirm that distinct 14C-labelled peptides are generated from these two species. These proteinchemical data supplement recent immunological findings, which demonstrate that component X is not a proteolytic fragment of the larger lipoyl acetyltransferase ( M , 70000) subunit.Incubation of the native PDC in the presence of [2-14C]pyruvate leads to rapid uptake of radiolabel, presumably as acetyl groups, into both E2 and protein X. Specific incorporation of acetyl groups declines to a similar extent on both polypeptides after inhibiting pyruvate dehydrogenase (El) activity by phosphorylation or omitting thiamine diphosphate (TPP) from the assay mixture. Addition of CoASH promotes the parallel deacetyhtion of both lipoyl acetyltransferase and protein X in a reaction which displays sensitivity to N-ethylmaleimide.The unique structural, enzymological and regulatory features of the mammalian pyruvate dehydrogenase complex (PDC) have prompted detailed investigation of its properties over many years [I, 21. The macromolecular array of three individual enzymes is responsible for controlling the supply of CoASAc to the tricarboxylic acid cycle by catalysing its formation from pyruvate. Glucose is conserved in mammals in starvation and diabetes by diminished flow through the mitochondria1 PDC [3]. Its overall activity is regulated by a phosphorylation/dephosphorylation system involving a specific pyruvate dehydrogenase kinase and a phosphatase, with phosphorylation resulting in inactivation [I 1.The major enzymatic activities, catalyzing discrete partial reactions of the complete process, have long been recognised as they can be purified readily in active form after dissociation of the complex under mild conditions [4, 51. The initial step involves decarboxylation of the substrate by pyruvate dehydrogenase (El), a TPP-requiring a2/32 tetramer, which also transfers two carbon units to lipoate residues, situated on lipoyl acetyltransferase (E2), in a reductive acetylation reaccorrespondence to

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