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 decarboxyase complex. Electrospray ionization-mass spectrometric analysis of E3 and the L-protein gave exactly the same molecular mass of 49753±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.
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January 1996
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Research Article|
January 01 1996
Glycine decarboxylase and pyruvate dehydrogenase complexes share the same dihydrolipoamide dehydrogenase in pea leaf mitochondria: evidence from mass spectrometry and primary-structure analysis Available to Purchase
Jacques BOURGUIGNON;
Jacques BOURGUIGNON
§
*Laboratoire de Physiologie Cellulaire Végétale, URA CNRS No.576, Département de Biologie Structurale et Moléculaire, CEA/Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France
§To whom correspondence and reprint requests should be addressed.
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Véronique MERAND;
Véronique MERAND
†Laboratoire de Spectrométrie de Masse des Protéines, CEA-CNRS, Institut de Biologie Structurale, 41 rue des Martyrs, 38027 Grenoble cedex 1, France
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Stephen RAWSTHORNE;
Stephen RAWSTHORNE
‡Brassica and Oilseeds Research Department, John Innes Centre, Colney, Norwich NR4 7UH, East Anglia, U.K.
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Eric FOREST;
Eric FOREST
†Laboratoire de Spectrométrie de Masse des Protéines, CEA-CNRS, Institut de Biologie Structurale, 41 rue des Martyrs, 38027 Grenoble cedex 1, France
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Roland DOUCE
Roland DOUCE
*Laboratoire de Physiologie Cellulaire Végétale, URA CNRS No.576, Département de Biologie Structurale et Moléculaire, CEA/Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France
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Publisher: Portland Press Ltd
Received:
July 06 1995
Revision Received:
September 01 1995
Accepted:
September 01 1995
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 313 (1): 229–234.
Article history
Received:
July 06 1995
Revision Received:
September 01 1995
Accepted:
September 01 1995
Citation
Jacques BOURGUIGNON, Véronique MERAND, Stephen RAWSTHORNE, Eric FOREST, Roland DOUCE; Glycine decarboxylase and pyruvate dehydrogenase complexes share the same dihydrolipoamide dehydrogenase in pea leaf mitochondria: evidence from mass spectrometry and primary-structure analysis. Biochem J 1 January 1996; 313 (1): 229–234. doi: https://doi.org/10.1042/bj3130229
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