Separation and characterization of aconitate hydratase isoenzymes from pig tissues

Eanes, Robert Z.; Kun, Ernest

doi:10.1016/0005-2744(71)90181-1

Cited by 33 publications

(13 citation statements)

References 12 publications

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“…These IRP-like proteins were found to encode mitochondrial ACO (Figures 2 and 4), a result consistent with studies that found the three ACO proteins in the mitochondrial proteome [35]. In animals, mitochondrial and cytosolic ACOs are encoded by distinct genes [36,37]. It is not the case in Saccharomyces cerevisiae, in which a unique gene encodes both mitochondrial and cytosolic ACOs [38,39].…”

Section: Discussionsupporting

confidence: 87%

The iron-responsive element (IRE)/iron-regulatory protein 1 (IRP1)–cytosolic aconitase iron-regulatory switch does not operate in plants

Arnaud

Ravet

Borlotti

et al. 2007

Biochemical Journal

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Animal cytosolic ACO (aconitase) and bacteria ACO are able to switch to RNA-binding proteins [IRPs (iron-regulatory proteins)], thereby playing a key role in the regulation of iron homoeostasis. In the model plant Arabidopsis thaliana, we have identified three IRP1 homologues, named ACO1-3. To determine whether or not they may encode functional IRP proteins and regulate iron homoeostasis in plants, we have isolated loss-of-function mutants in the three genes. The aco1-1 and aco3-1 mutants show a clear decrease in cytosolic ACO activity. However, none of the mutants is affected in respect of the accumulation of the ferritin transcript or protein in response to iron excess. cis-acting elements potentially able to bind to the IRP have been searched for in silico in the Arabidopsis genome. They appear to be very rare sequences, found in the 5 -UTR (5 -untranslated region) or 3 -UTR of a few genes unrelated to iron metabolism. They are therefore unlikely to play a functional role in the regulation of iron homoeostasis. Taken together, our results demonstrate that, in plants, the cytosolic ACO is not converted into an IRP and does not regulate iron homoeostasis. In contrast with animals, the RNA binding activity of plant ACO, if any, would be more likely to be attributable to a structural element, rather than to a canonical sequence.

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Section: Discussionsupporting

confidence: 87%

The iron-responsive element (IRE)/iron-regulatory protein 1 (IRP1)–cytosolic aconitase iron-regulatory switch does not operate in plants

Arnaud

Ravet

Borlotti

et al. 2007

Biochemical Journal

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“…For the preparation of plastids, intact protoplasts resuspended in basic medium were passed through a fine nylon mesh as described above. The broken protoplasts were centrifuged for 10,500 rpm (10,OOOg) for 100 min. At the conclusion of this step, a continuous gradient of Percoll was obtained in each tube (24).…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Cytosolic Aconitase in Higher Plant Cells

Brouquisse¹,

Nishimura²,

Gaillard³

et al. 1987

Plant Physiol.

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ABSTRACFProtoplasts obtained from sycamore (Acer pseadplUatw) cell suspensions were found to be highly intact. If the protoplasts were taken up and expelled through a fine nylon mesh, all the protoplasts were ruptured leaving the fragile amyloplasts lagely intact. Aconitase hydratase ( for 1 h resulted in a final yield of protoplasts near 100%. After digestion, the suspension was filtered through one layer of Miracloth (Krantex, Alfortville, France) that retained any undigested cell aggregates, and protoplasts were collected by centrifuging at 150g for 10 min and then washed twice with 50 ml of culture medium containing 0.5 M mannitol (suspension medium). Protoplasts were stored in suspension medium and were normally used within 2 to 3 h.Gentle Rupture of Protoplasts and Separation of the Organelles from the Cytosolic Fraction. Since sycamore cell protoplasts have an average diameter of 20 to 30 gm, a rapid and effective procedure for the gentle rupture of intact protoplasts (i.e. for stripping the cell membrane) is to pass protoplasts through a fine nylon mesh (20 ,um) affixed to the cut end of a 25-ml disposable syringe (20). Thus, protoplasts (equivalent to 20.106 cells) are pelleted after dilution with suspending medium and resuspended in about 5 ml of a basic medium: 0.5 M mannitol; 0.1% (w/v) BSA; 1% (w/v) insoluble polyvinylpyrrolidone (Kca 25, Serva); 5 mM DTT; 10 Mm leupeptin; 5 mm phosphate buffer (pH 7.4); these last parameters were chosen because they correspond to the phosphate concentration and the pH of the cytosolic compartment previously determined by 31P nuclear magnetic resonance studies (22). If the protoplasts are taken up and expelled through the 20 Mm nylon mesh two times, they will be completely ruptured. In order to separate the cytosolic fraction from the cell organelles, we subjected the broken protoplast fraction to centrifugation to yield a pellet largely free of cytosol and a supernatant enriched in cytosolic enzymes. In order to prevent starch grains from tearing through the plastid envelope, the centrifugal g force was gradually increased by using an automatic rate controller (Sorvall) and centrifugation was carried out in three steps (100g for 5 min, 500g for 5 min, and 12,000g for 10 min; Sorvall rotor SS34). Each supernatant was centrifuged in a new tube, and the three successive pellets were combined together. This procedure ruptures all the protoplasts leaving the fragile amyloplasts and mitochondria largely intact (17).Isolation of Amyloplastids. For the preparation of plastids, intact protoplasts resuspended in basic medium were passed through a fine nylon mesh as described above.

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“…Thus under conditions of low iron, the level of transferrin receptor rises while the level of intracellular ferritin falls, leading to an increase in intracellular free iron. Analysis of animal cytosolic and mt aconitase isoforms has revealed that these proteins have different biochemical properties and are encoded by distinct genes (Eanes and Kun 1971;Povey et al 1976;Slaughter et al 1978;Kennedy et al 1992). By contrast, plant mt and cytosolic aconitases share similar kinetic properties (Brouquisse et al 1987;De Bellis et al 1993) and growing evidence suggests that they are encoded by the same gene(s).…”

Section: Introductionmentioning

confidence: 94%

Aconitase plays a role in regulating resistance to oxidative stress and cell death in Arabidopsis and Nicotiana benthamiana

et al. 2006

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In animals, aconitase is a bifunctional protein. When an iron-sulfur cluster is present in its catalytic center, aconitase displays enzymatic activity; when this cluster is lost, it switches to an RNA-binding protein that regulates the translatability or stability of certain transcripts. To investigate the role of aconitase in plants, we assessed its ability to bind mRNA. Recombinant aconitase failed to bind an iron responsive element (IRE) from the human ferritin gene. However, it bound the 5' UTR of the Arabidopsis chloroplastic CuZn superoxide dismutase 2 (CSD2) mRNA, and this binding was specific. Arabidopsis aconitase knockout (KO) plants were found to have significantly less chlorosis after treatment with the superoxide-generating compound, paraquat. This phenotype correlated with delayed induction of the antioxidant gene GST1, suggesting that these KO lines are more tolerant to oxidative stress. Increased levels of CSD2 mRNAs were observed in the KO lines, although the level of CSD2 protein was not affected. Virus-induced gene silencing (VIGS) of aconitase in Nicotiana benthamiana caused a 90% reduction in aconitase activity, stunting, spontaneous necrotic lesions, and increased resistance to paraquat. The silenced plants also had less cell death after transient co-expression of the AvrPto and Pto proteins or the pro-apoptotic protein Bax. Following inoculation with Pseudomonas syringae pv. tabaci carrying avrPto, aconitase-silenced N. benthamiana plants expressing the Pto transgene displayed a delayed hypersensitive response (HR) and supported higher levels of bacterial growth. Disease-associated cell death in N. benthamiana inoculated with P. s. pv. tabaci was also reduced. Taken together, these results suggest that aconitase plays a role in mediating oxidative stress and regulating cell death.

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Separation and characterization of aconitate hydratase isoenzymes from pig tissues

Cited by 33 publications

References 12 publications

The iron-responsive element (IRE)/iron-regulatory protein 1 (IRP1)–cytosolic aconitase iron-regulatory switch does not operate in plants

The iron-responsive element (IRE)/iron-regulatory protein 1 (IRP1)–cytosolic aconitase iron-regulatory switch does not operate in plants

Characterization of a Cytosolic Aconitase in Higher Plant Cells

Aconitase plays a role in regulating resistance to oxidative stress and cell death in Arabidopsis and Nicotiana benthamiana

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