Pyruvate dehydrogenase complex from the primitive insect trypanosomatid, Crithidia fasciculata: dihydrolipoyl dehydrogenase-binding protein has multiple lipoyl domains.
The pyruvate dehydrogenase complex (PDC) has been purified to apparent homogeneity from the insect trypanosomatid, Crithidia fasciculata, a member of the most primitive eukaryotic group to contain mitochondria. Separation of the purified PDC by SDS-PAGE yielded five bands of 70 (p70), 60 (p60), 55, 46 and 36.5 kDa, which appeared to correspond to dihydrolipoyl dehydrogenase binding protein (E3BP), dihydrolipoyl transacetylase (E2), E3, E1 alpha and E1 beta, respectively. The purified complex did not exhibit endogenous PDHa kinase activity. p70 was much less abundant than p60. Polyclonal antisera raised against p70 did not cross-react with p60, and antisera raised against p60 did not cross-react with p70, suggesting that p60 did not arise from p70 by proteolysis. Both p70 and p60 contained similar amino terminal sequences. Both sequences contained the MPALSP motif similar to sequences present in both E3BP and E2 from other sources. Incubation of the purified PDC with [2-14C]pyruvate in the absence of CoA resulted in the acetylation of both p70 and p60, suggesting that both proteins contained lipoyl domains, but the specific incorporation of label into p70 was significantly greater than for p60. Limited proteolysis of the acetylated complex with trypsin yielded two major fragments derived from p60 of 35 and 30 kDa, corresponding to E2L and E2I, and one major acetylated fragment of 58 kDa derived from p70. Therefore, these results suggest that p70 is an E3BP and given its apparent M(r) and degree of acetylation, it contains multiple lipoyl domains.
Diaz
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F
Department of Biology, University of Toledo, OH 43606-3390, USA. FDIAZ@uoft02.utoledo.edu
Komuniecki
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R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D016831:Crithidia fasciculata / Q000201:enzymology*; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007313:Insecta / Q000469:parasitology; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology; D008929:Mitochondria, Heart / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011092:Polyethylene Glycols; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014357:Trypsin; D014461:Ultracentrifugation
D046911:Macromolecular Substances; C489060:PDX1 protein, S cerevisiae; D010446:Peptide Fragments; D010455:Peptides; D011768:Pyruvate Dehydrogenase Complex; D029701:Saccharomyces cerevisiae Proteins; C489061:pyruvate dehydrogenase complex component X, Bos taurus; D011092:Polyethylene Glycols; D008058:Dihydrolipoamide Dehydrogenase; D014357:Trypsin