Inactivation of the glyoxysomal citrate synthase from castor bean endosperm by 5,5?-dithiobis(2-nitrobenzoic acid) (DTNB)

Schnarrenberger, Claus; Fitting, K. H.; Tetour, M.; Zehler, Heinz

doi:10.1007/bf01276275

Cited by 11 publications

(5 citation statements)

References 22 publications

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“…Both mutants had reduced CSY activity at days 0 to 2 relative to the wild type, and although this difference is small, it is seen consistently. It is reported that 5,59-dithio-bis (2-nitrobenzoic acid) inhibits peroxisomal CSY activity but not mitochondrial CSY in castor bean (Ricinus communis) endosperm (Schnarrenberger et al, 1980), but we could not reliably distinguish these enzymes in Arabidopsis using 5,59-dithiobis(2-nitrobenzoic acid) (data not shown).…”

Section: Isolation and Characterization Of Csy Knockout Mutantsmentioning

confidence: 64%

Arabidopsis Peroxisomal Citrate Synthase Is Required for Fatty Acid Respiration and Seed Germination

2005

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We tested the hypothesis that peroxisomal citrate synthase (CSY) is required for carbon transfer from peroxisomes to mitochondria during respiration of triacylglycerol in Arabidopsis thaliana seedlings. Two genes encoding peroxisomal CSY are expressed in Arabidopsis seedlings, and seeds from plants with both CSY genes disrupted were dormant and did not metabolize triacylglycerol. Germination was achieved by removing the seed coat and supplying sucrose, but the seedlings still did not use triacylglycerol. The mutant seedlings were resistant to 2,4-dichlorophenoxybutyric acid, indicating a block in peroxisomal b-oxidation, and were unable to develop further after transfer to soil. The mutant phenotype was complemented with a cDNA encoding CSY with either its native peroxisomal targeting sequence (PTS2) or a heterologous PTS1 sequence from pumpkin (Cucurbita pepo) malate synthase. These results suggest that peroxisomal CSY in Arabidopsis is not only a key enzyme of the glyoxylate cycle but also catalyzes an essential step in the respiration of fatty acids. We conclude that citrate is exported from the peroxisome during fatty acid respiration, whereas in yeast, acetylcarnitine is exported.

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Section: Isolation and Characterization Of Csy Knockout Mutantsmentioning

confidence: 64%

Arabidopsis Peroxisomal Citrate Synthase Is Required for Fatty Acid Respiration and Seed Germination

2005

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“…However, there are also a number of bacteria for which the molecular mass of the enzyme has been reported to be ≈ 280 kDa or even more [46]. Many regulatory compounds [NADH, α‐oxoglutarate, 5,5′‐dithiobis‐(2‐nitrobenzoic acid), AMP, ATP, KCl, aggregation state] can influence the CS activity from various sources [46–48].…”

Section: Resultsmentioning

confidence: 99%

Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants

Schnarrenberger

Martin

2002

European Journal of Biochemistry

137

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The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for aminoacid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes. The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among a-proteobacteria, the group of eubacteria as de®ned under rRNA systematics from which mitochondria arose. Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from a-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution. Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants. The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their a-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not speci®cally a-proteobacterial, homologues. A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the a-proteobacterial ancestor of mitochondria to branch speci®cally with their homologues encoded in the genomes of contemporary a-proteobacteria. Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the a-proteobacterial ancestor of mitochondria should, in phylogenetic trees, branch with homologues that are no longer found in most a-proteobacterial genomes, and some should reside on long branches that reveal anity to eubacterial rather than archaebacterial homologues, but no particular anity for any speci®c eubacterial donor.

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“…6). The amino acid sequence of the mature enzyme was found to be about 40~o identical to the enzymes from gram-negative bacteria whereas it was only about 20 ~o identical to the enzymes from eukaryotic mitochondria, gCS has been reported to have structural and enzymatic properties distinct from those of mitochondrial enzymes, such as isoelectric point [44], ATPbinding properties [29,44] and inhibition by 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB) [30]. The ATP-binding and DTNB-inhibition properties of gCS resemble those of prokaryotic citrate synthases [44].…”

Section: Discussionmentioning

confidence: 99%

“…The reaction mixture contained 100 mM Tris-HC1 pH 8.0, 100 #M DTNB, 1 mM MgCI2, 1 mM oxaloacetate, and 200 #M acetylCoA in 1 ml. The reaction was started by adding a solution of enzyme to avoid inhibition of the enzyme by DTNB [30]. Catalase and cytochrome c oxidase were assayed as described previously [4].…”

Section: Assays Of Enzymatic Activities and Analysis Of Proteinsmentioning

confidence: 99%

Molecular characterization of a glyoxysomal citrate synthase that is synthesized as a precursor of higher molecular mass in pumpkin

et al. 1995

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A cDNA clone for glyoxysomal citrate synthase (gCS) was isolated from a lambda gt11 cDNA library prepared from etiolated pumpkin cotyledons. The cDNA of 1989 bp consisted of a 1548 bp open reading frame that encoded 516 amino acid residues. The deduced amino acid sequence of gCS did not have a typical peroxisomal targeting signal at its carboxyl terminal. A study of expression in vitro of the cDNA and an analysis of the amino-terminal sequence of the citrate synthase indicated that gCS is synthesized as a larger precursor that has a cleavable amino-terminal presequence of 43 amino acids. The predicted amino-terminal sequence of pumpkin gCS was highly homologous to those of other microbody enzymes, such as 3-ketoacyl-CoA thiolase of rat and malate dehydrogenase of watermelon that are also synthesized as precursors of higher molecular mass. Immunoblot analysis showed that the level of gCS protein increased markedly during germination and decreased rapidly during the light-induced transition of microbodies from glyoxysomes to leaf peroxisomes. By contrast, the level of mRNA for gCS reached a maximum earlier than that of the protein and declined even in darkness. The level of the mRNA was low during the microbody transition. These results indicate that the accumulation of the gCS protein does not correspond to that of the mRNA and that degradation of gCS is induced during the microbody transition, suggesting that post-transcriptional regulation plays an important role in the microbody transition.

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Inactivation of the glyoxysomal citrate synthase from castor bean endosperm by 5,5?-dithiobis(2-nitrobenzoic acid) (DTNB)

Cited by 11 publications

References 22 publications

Arabidopsis Peroxisomal Citrate Synthase Is Required for Fatty Acid Respiration and Seed Germination

Arabidopsis Peroxisomal Citrate Synthase Is Required for Fatty Acid Respiration and Seed Germination

Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants

Molecular characterization of a glyoxysomal citrate synthase that is synthesized as a precursor of higher molecular mass in pumpkin

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