Glycine decarboxylase and pyruvate dehydrogenase complexes share the same dihydrolipoamide dehydrogenase in pea leaf mitochondria: evidence from mass spectrometry and primary-structure analysis.
In order to compare the dihydrolipoamide dehydrogenase associated with the pyruvate dehydrogenase complex (E3) with that associated with the glycine decarboxylase complex (L-protein), we report for the first time the purification and characterization of the E3 component from pea leaf mitochondria. The first 30 amino acids of the N-terminal sequence of the mature E3 protein are identical with those of the mature L-protein of the glycine decarboxylase complex. Electrospray ionization-mass spectrometric analysis of E3 and the L-protein gave exactly the same molecular mass of 49,753 +/- 5 Da. We have also confirmed the primary structure of the L-protein, in particular the C-terminal sequence, deduced from the cDNA published by Bourguignon, Macherel, Neuburger and Douce [(1992) Eur. J. Biochem. 204, 865-873]. Western-blot analysis shows that specific polyclonal antibodies raised against the L-protein recognize specifically both E3 and L-protein but not the porcine dihydrolipoamide dehydrogenase. We conclude that, in pea leaf mitochondria, the pyruvate dehydrogenase and glycine decarboxylase complexes share the same dihydrolipoamide dehydrogenase. We have also confirmed by MS analysis that the FAD is not covalently bound to the enzyme.
Bourguignon
J
J
URA CNRS No 576, Département de Biologie Structurale et Moléculaire, CEA/Grenoble, France.
Merand
V
V
Rawsthorne
S
S
Forest
E
E
Douce
R
R
D000594:Amino Acid Oxidoreductases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D050959:Glycine Decarboxylase Complex; D050841:Glycine Dehydrogenase (Decarboxylating); D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology* / Q000648:ultrastructure; D018515:Plant Leaves / Q000201:enzymology / Q000648:ultrastructure; D010940:Plant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D010940:Plant Proteins; D011768:Pyruvate Dehydrogenase Complex; D000594:Amino Acid Oxidoreductases; D050959:Glycine Decarboxylase Complex; D050841:Glycine Dehydrogenase (Decarboxylating); D008058:Dihydrolipoamide Dehydrogenase