Glyoxysomal malate-dehydrogenase and malate synthase from Soybean cotyledons (Glycine max. L.): Enzyme association, antibody-production and cDNA cloning

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Etat: Serval
Version: de l'auteur
ID Serval
serval:BIB_0B65EB7705F7
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Glyoxysomal malate-dehydrogenase and malate synthase from Soybean cotyledons (Glycine max. L.): Enzyme association, antibody-production and cDNA cloning
Périodique
Planta
Auteur(s)
Guex N., Henry H., Flach J., Richter H., Widmer F.
ISSN
0032-0935
Statut éditorial
Publié
Date de publication
1995
Peer-reviewed
Oui
Volume
197
Numéro
2
Pages
369-375
Langue
anglais
Résumé
In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD(+) oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M(r)) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M, 510 000 and of gMDH as a dimer of M, 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X(6))HL motif also found in plant and mammalian thiolases.
Mots-clé
ENZYME COMPLEX, GLYCINE, GLYOXYSOME, MALATE DEHYDROGENASE, MALATE SYNTHASE, PEROXISOMAL 3-KETOACYL-COA THIOLASE, SACCHAROMYCES-CEREVISIAE, 3-OXOACYL-COA THIOLASE, TERMINAL PRESEQUENCE, ISOCITRATE LYASE, GLYOXYLATE CYCLE, MESSENGER-RNA, RAT-LIVER, COMPLEXES, SEQUENCE
Pubmed
Web of science
Création de la notice
13/08/2015 9:01
Dernière modification de la notice
03/03/2018 13:36
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