Salt-mediated interconversions and purification of malate synthase from germinating soybean cotyledons (Glycine max. L.)

Henry, Hughes; Escher, Claire-Lise; Widmer, François

doi:10.1016/0168-9452(92)90004-6

Cited by 8 publications

(18 citation statements)

References 26 publications

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“…This is in contrast to what has been described for other malate synthases such as the synthases from germinating soybean cotyledons (Glycine max. L.), which may be dimeric or decameric aggregates depending on the ionic strength [28], or the malate synthase from S. cerevisiae, which has been described as a homotrimeric protein [29]. Our determination of molecular mass of the crude extract enzyme shows that aggregation of subunits, which could be dissociated in the purification process (heat treatment, salt concentration) does not occur in our case.…”

Section: Discussionmentioning

confidence: 73%

Molecular Characterization of Escherichia coli Malate Synthase G

Molina

Pellicer

Badı́a

et al. 1994

European Journal of Biochemistry

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Two genes encoding the enzymes malate synthase G and glycolate oxidase, have been linked to locus glc (64.5 min), responsible for glycolate utilization in Escherichia coli. The gene encoding malate synthase G, for which we propose the notation glcB, has been cloned, sequenced and found to correspond to a 2262-nucleotide open-reading frame, which can encode a 723-amino-acid polypeptide, clearly different from the isoenzyme malate synthase A, which has 533 amino acids. Northern-blot experiments indicate that glcB was expressed as an apparently monocistronic transcript, inducible by glycolate. Malate synthase G was purified to near homogeneity. The molecular mass determined by gel filtration yielded a value of 82 kDa for the purified enzyme and the same value as for the crude extract enzyme, indicating a monomeric structure. Despite the lower sequence similarity between malate synthase G and the other reported malate synthases, three out of nine consensus boxes defined in most of these enzymes are conserved in addition to a cysteine residue that has been reported to be important for the catalytic mechanisms.Two isoenzymes of malate synthase have been described for the metabolism of glyoxylate in Escherichia coli. Malate synthase A (MSA), involved in the glyoxylate by-pass reaction, is encoded by the gene aceB belonging to the ace operon located at 91 min on the genetic map [l], and is predominant (60% total activity) in cells metabolizing the glyoxylate formed in the dissimilation of acetate [2]. The other isoenzyme, malate synthase G (MSG), encoded by a gene mapped in the locus glc, accounts for almost the entire malate-synthesizing activity in cells metabolizing the glyoxylate formed during growth on glycolate [2]. Both isoenzymes catalyze the condensation of glyoxylate and acetyl-CoA to yield malate, display very similar kinetic parameters for the substrate (31, and are distinguished on the basis of their chromatographic separation, thermostability and sensitivity to inhibitors such as glycolate and oxalate [3].In glycolate metabolism, this compound is first oxidized to glyoxylate by the action of glycolate oxidase [4, 51. Glyoxylate is subsequently metabolized by two divergent codensation reactions; one reaction is catalyzed by glyoxylate carboligase, which converts two glyoxylate molecules to tartronic semialdehyde and carbon dioxide [6], and the other reaction is catalyzed by MSG. The latter process can be considered a reaction of an oxidative pathway if glyoxylate is metabolized through the dicarboxylic cycle, or a reaction of Correspondence to L. Baldomi,

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Section: Discussionmentioning

confidence: 73%

Molecular Characterization of Escherichia coli Malate Synthase G

Molina

Pellicer

Badı́a

et al. 1994

European Journal of Biochemistry

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“…of 23 nkat/mg protein and a yield of 50%. In comparison, precipitation with (NH4)2SO 4 generally used in the purification protocols results in a specific activity of only 5.1 nkat/mg protein for preparations from rape seed cotyledons [7], 7.3 nkat/mg protein for soybean cotyledons [8] and 10.8 nkat/mg protein for cotton cotyledons. The final purification step of our procedure is a sedimentation centrifugation step which provides a good yield and high specific activity of malate synthase.…”

Section: Resultsmentioning

confidence: 99%

“…The enzyme was purified from several plant species [4][5][6][7][8]. Its activity is strictly dependent on divalent cations, such as Mg 2+ and it is comprised of subunits with a molecular weight of around 63 kDa which readily aggregate to oligomeric complexes [6][7][8][9]. The aggregation behavior of malate synthase can be used for enzyme purification.…”

Section: Introductionmentioning

confidence: 99%

Rapid purification of malate synthase from cotyledons of Brassica napus L

Hoppe

Theimer

1995

FEBS Letters

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A rapid and efficient method for the purifcation of malate synthase, an enzyme uniquely confined to glyoxysomes, from cotyledons of Brassica napus L. has been developed. The two step purification procedure is based on the consequent utilization of the tendency of malate synthase to form high molecular weight aggregates. Malate synthase was purified 75-fold to apparent homogeneity with a specific activity of 180 nkaffmg protein. The estimated molecular weight of malate synthase subunits was 63 kDa. Polyclonal antibodies raised against malate synthase in rabbits detect on Western blots only one single polypeptide with an identical molecular weight.

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“…A DEAE-Sepharose CL-6B column (diameter = 1.6 cm; flow rate = 0.5 mlmin-1) containing 60 ml of anion-exchange gel (Pharmacia) was used to recover nonadsorbed MS and gMDH after equilibration at pH 9.0 in high-ionic-strength buffer (25 mM Tris-HC1 buffer containing 5 mM MgCI z and 50 mM KC1; Henry et al 1992).…”

Section: Diethylaminoethyl (Deae)-sepharose Cl-6b Anion Exchangementioning

confidence: 99%

“…It has been shown that MS from soybean cotyledons can undergo structural interconversions depending on ionic strength (Henry et al 1992), and that MS obtained from cucumber seeds can be found as monomeric (5S), octameric (19S; predominant form) or multimeric (100S) species. This high-relativemolecular-mass (Mr) species was often recovered in the microsomal fraction of sucrose gradients, and was therefore long believed to be an MS precursor synthesized in the endoplasmic reticulum (for a review, see Beevers 1979).…”

Section: Introductionmentioning

confidence: 99%

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

Guex

Henry

Flach

et al. 1995

Planta

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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 (Mr) of 670,000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M(r) 510,000 and of gMDH as a dimer of M(r) 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(X6)HL motif also found in plant and mammalian thiolases.

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Salt-mediated interconversions and purification of malate synthase from germinating soybean cotyledons (Glycine max. L.)

Cited by 8 publications

References 26 publications

Molecular Characterization of Escherichia coli Malate Synthase G

Molecular Characterization of Escherichia coli Malate Synthase G

Rapid purification of malate synthase from cotyledons of Brassica napus L

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

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