The pyruvate dehydrogenase complex from the parasitic nematode Ascaris suum: novel subunit composition and domain structure of the dihydrolipoyl transacetylase component.
The pyruvate dehydrogenase complex (PDC) from muscle of the adult parasitic nematode Ascaris suum plays a unique role in its anaerobic mitochondrial metabolism. Resolution of the intact complex in high salt dissociates the pyruvate dehydrogenase subunit but leaves the dihydrolipoyl dehydrogenase subunit (E3) and two other proteins with apparent M(r)s of 45 and 43 kDa bound to the dihydrolipoyl transacetylase (E2) core. These proteins are not observable on Coomassie brilliant blue-stained gels of other eukaryotic PDCs, but the 45-kDa protein is similar in apparent M(r), pI, and sensitivity to trypsin to the Kb subunit of the bovine kidney PDH alpha kinase. Acetylation of the ascarid PDC with [2-14C]pyruvate under conditions designed to maximize the incorporation of label into protein yielded only a single radiolabeled subunit, E2. These results confirm earlier reports that the ascarid PDC lacks protein X, an integral component recently identified in other eukaryotic PDCs. About 1.6 to 1.8 mol of 14C was incorporated/mole of E2, suggesting that the ascarid E2 contained two lipoly-bearing domains. Domain mapping of the 14C-acetylated ascarid E2 by limited tryptic digestion identified two lipoyl-bearing fragments with apparent M(r)s of 50 and 34 kDa and two core fragments with apparent M(r)s of 46 and 30 kDa. The ascarid E2 domain structure appears to be similar to that of other E2s. However, it appears that the subunit-binding domain (E2B) of the ascarid E2 may be significantly larger or be flanked by larger than normal interdomain regions. An enlarged E2B domain may be necessary to accommodate the additional binding of E3 to the E2 subunit in the ascarid complex, in the absence of protein X.
Komuniecki
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R
Department of Biology, University of Toledo, Ohio 43606.
Rhee
R
R
Bhat
D
D
Duran
E
E
Sidawy
E
E
Song
H
H
D000123:Acetyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D002417:Cattle; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D007106:Immune Sera; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D000939:Epitopes; D007106:Immune Sera; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011768:Pyruvate Dehydrogenase Complex; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase