Glyoxysomal malate dehydrogenase and malate synthase from soybean cotyledons (Glycine maxL.): enzyme association, antibody production and cDNA cloning
✍ Scribed by Nicolas Guex; Hugues Henry; Jean Flach; Hannes Richter; François Widmer
- Publisher
- Springer-Verlag
- Year
- 1995
- Tongue
- English
- Weight
- 899 KB
- Volume
- 197
- Category
- Article
- ISSN
- 0032-0935
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✦ Synopsis
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-ionicstrength 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 (Mr) of 670000. Dissociation was achieved in the presence of 50 mM KC1 and 5 mM MgC12, with the elution of MS as an octamer of M r 510 000 and of gMDH as a dimer of Mr 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 k 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