Inactivation of the tricarboxylate carrier of liver mitochondria by (−)-erythrofluorocitrate

Eanes, Robert Z.; Skilleter, David N.; Kun, Ernest

doi:10.1016/0006-291x(72)90794-2

Cited by 24 publications

(10 citation statements)

References 7 publications

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“…with intramitochondrial generation of citric acid, were inhibited to an equal degree (Eanes et al, 1972;Brand et al, 1973;Eanes and Kun, 1974). Since aconitase is not involved directly in triglyceride metabolism, the existence of a (−)-erythro-fluorocitrate inhibitory site hindering citrate transport through the inner mitochondrial membrane was suggested (Eanes et al, 1972;Kirsten et al, 1978).…”

Section: Pathogenesis: Physiological and Biochemical Features Of Fa Amentioning

confidence: 93%

“…Since aconitase is not involved directly in triglyceride metabolism, the existence of a (−)-erythro-fluorocitrate inhibitory site hindering citrate transport through the inner mitochondrial membrane was suggested (Eanes et al, 1972;Kirsten et al, 1978).…”

Section: Pathogenesis: Physiological and Biochemical Features Of Fa Amentioning

confidence: 94%

“…The biochemical and physiological effects of FA include: excessive citrate accumulation (Buffa and Peters, 1950;Egyed and Brisk, 1965;Buffa et al, 1973); disturbed citrate transport in mitochondria (Eanes et al, 1972;Kirsten et al, 1978); increased lactate level (Engel et al, 1954;Schultz et al, 1982;Taitelman et al, 1983b); deviations in carbohydrate metabolism regulation (Elliott and Phillips, 1954;Bobyleva-Guarriero et al, 1984); decreased level of free fatty acids (Liang, 1977); decreased tissue level of macroergotic compounds (Williamson, 1967;Stewart et al, 1970); increased adenosine level (Liang, 1977); decreased oxygen consumption (Saito, 1990); disturbed balance of bivalent cations, specifically Ca 2+ (Buffa and Peters, 1950;Bosakowski and Levin, 1986); acid-base imbalance (Taitelman et al, 1983b;Szerb and Redondo, 1993); increased ammonium level (Stewart et al, 1970); changed γ -aminobutyric acid level (Maytnert and Kaji, 1962;Stewart et al, 1970); increased plasma levels of phosphorus and various enzymes (Bgin et al, 1972); hemodynamic disorders (Liang, 1977).…”

Section: Pathogenesis: Physiological and Biochemical Features Of Fa Amentioning

confidence: 99%

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Toxicology of fluoroacetate: a review, with possible directions for therapy research

2006

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Fluoroacetate (FA; CH2FCOOR) is highly toxic towards humans and other mammals through inhibition of the enzyme aconitase in the tricarboxylic acid cycle, caused by 'lethal synthesis' of an isomer of fluorocitrate (FC). FA is found in a range of plant species and their ingestion can cause the death of ruminant animals. Some fluorinated compounds -- used as anticancer agents, narcotic analgesics, pesticides or industrial chemicals -- metabolize to FA as intermediate products. The chemical characteristics of FA and the clinical signs of intoxication warrant the re-evaluation of the toxic danger of FA and renewed efforts in the search for effective therapeutic means. Antidotal therapy for FA intoxication has been aimed at preventing fluorocitrate synthesis and aconitase blockade in mitochondria, and at providing citrate outflow from this organelle. Despite a greatly improved understanding of the biochemical mechanism of FA toxicity, ethanol, if taken immediately after the poisoning, has been the most acceptable antidote for the past six decades. This review deals with the clinical signs and physiological and biochemical mechanisms of FA intoxication to provide an explanation of why, even after decades of investigation, has no effective therapy to FA intoxication been elaborated. An apparent lack of integrated toxicological studies is viewed as a limiter of progress in this regard. Two principal ways of developing effective therapies for FA intoxication are considered. Firstly, competitive inhibition of FA interaction with CoA and of FC interaction with aconitase. Secondly, channeling the alternative metabolic pathways by orienting the fate of citrate via cytosolic aconitase, and by maintaining the flux of reducing equivalents into the TCA cycle via glutamate dehydrogenase.

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Section: Pathogenesis: Physiological and Biochemical Features Of Fa Amentioning

confidence: 93%

Section: Pathogenesis: Physiological and Biochemical Features Of Fa Amentioning

confidence: 94%

Section: Pathogenesis: Physiological and Biochemical Features Of Fa Amentioning

confidence: 99%

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Toxicology of fluoroacetate: a review, with possible directions for therapy research

2006

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“…Alternatively the central carboxyl group of fluorocitrate may hydrogen-bond to the hydrogen abstracting site. Further biochemical studies on this problem and on the effect of fluorocitrate on the tricarboxylate carrier of mitochondria (Lanes et al, 1972) are now in progress in various laboratories.…”

Section: Rubidium Ammonium Hydrogen Fluorocitrate Dihydratementioning

confidence: 99%

The crystal structure of rubidium ammonium hydrogen fluorocitrate dihydrate

Carrell¹,

Glusker²

1973

Acta Crystallogr Sect B

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The crystal structure of the racemate of rubidium ammonium hydrogen fluorocitrate dihydrate, RbNHaH(C6HaO7F).2H20, containing the isomer of fluorocitrate that inhibits the enzyme aconitase, has been determined. The configurations of the two isomers in the racemate are I R:2R and 1S:2S for 1-fluoro-2-hydroxy-l,2,3-propanetricarboxylic acid. The crystals are triclinic, space group PT, Z=2, with unit-cell dimensions a=8-458 (2), b= 10.910 (3), c=7.516 (2) A,, 0c= 102.80 ° (3), fl= 115.90 ° (3), 7,=80-50 ° (4). The observed and calculated densities are 1.92 and 1.925 g cm -3 respectively. The structure was solved from the Patterson map and refined to an R value of 0.072. All hydrogen atoms, except that on a carboxyl group of the anion, were located from a difference electron-density map. The fluorocitrate ion forms a tridentate chelate with a rubidium ion, the fluorine atom being shared by two rubidium coordination polyhedra. There is disorder of rubidium ions with ammonium ions to an approximate extent of 84 % and, corresponding to this, the fluorocitrate ion, also disordered to the extent of 84%, is presumed to pack so that in each case the fluorine atom is coordinated to the rubidium ion.

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“…We have previously identified the biologically active species of the four possible isomers of monofluorocitric acid as (-)erythrofluorocitrate (1, 2); its structure subsequently was confirmed by x-ray crystallography (3,4). Kinetic evidence indicated that (-)erythrofluorocitrate, when preincubated with isolated mitochondria at concentrations of pmol of fluorocitrate per mg of mitochondrial protein, irreversibly inhibited bidirectional citrate transport (5)(6)(7)(8). Whereas the high sensitivity of citrate influx to low concentrations of fluorocitrate in intact mitochondria has been confirmed (9), an unusually low competitive ki value for fluorocitrate was also reported for the aconitase activity of disrupted mitochondria that had been preincubated with 1 mM Mg2+ (9).…”

mentioning

confidence: 97%

Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate

Kun

Kirsten

Sharma

1977

Proc. Natl. Acad. Sci. U.S.A.

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A soluble extract of the mitochondrial compartment composed of the inner membrane and matrix catalyzes the enzymatic synthesis and hydrolysis of the 1:1 adduct of citric acid and glutathione. (9) was also explained by the stringent requirement of preincubation of mitochondria with fluorocitrate in the absence of millimolar concentrations of citrate (cf. 7, 8), allowing for the formation of two protein-fluorocitrate thioesters (8).The present report is concerned with the identification of a mitochondrial enzyme system that catalyzes the formation of the 1:1 adduct of glutathione and citric acid. The adduct was identified as the thioester of glutathione, and kinetic evidence was also obtained that the mitochondrial extract contained, in addition to the thioester-forming enzyme system, a hydrolase capable of degrading the thioester. The citryl-glutathioneforming enzyme system proved to be the most sensitive catalytic component of mitochondria towards the irreversible inhibitory action of (-)erythrofluorocitrate, suggesting that the molecular site of action of this highly toxic agent has been identified. METHODSLysosome-free mitochondria and mitoplasts (defined as the matrix surrounded by the inner membrane) were isolated from rat livers as described (12). An extract of mitoplasts was prepared by stirring (at 00) of a suspension of mitoplasts (30 mg of protein per ml) in 0.25 M sucrose/mannitol medium (12) containing 1 mg of Brij 56 (Sigma Chemicals) per 10 mg of mitoplast protein for 15 min. The resulting lysate was diluted 1:3 with the medium; after removal of large granules (unbroken mitoplasts) by centrifugation at 10,000 X g (10 min at 40), the supernatant (containing soluble extract of the inner membrane and soluble proteins of the matrix and inner membrane vesicles) was subjected to ultracentrifugation (145,000 X g for 1 hr at 20). The clear supernatant was concentrated by centrifugation (at 20) in Amicon CF-25 membrane cones at 100 X g to one-fourth of the original volume. Approximately 250 mg of protein extract was obtained by this technique from 500 mg (protein) of mitoplast starting material. The concentrated extract was used directly in most experiments, unless specified, whereas the sediment containing the inner membrane vesicles was rehomogenized in the suspending medium. Both fractions were stored at -15°until used. The enzymatic activity of the soluble extract decayed slowly at -150 (about half of the activity was lost in 2 weeks) but much more rapidly after repeated thawing and refreezing. The activity was completely recovered when the stored extract was preincubated for 5-10 min with 5 mM reduced glutathione (GSH), demonstrating the essential role Abbreviations: GSH, reduced glutathione; GSSG, oxidized glutathione; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; citryl-SG and malyl-SG, glutathione thioesters of citric and malic acids, respectively. 4942

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Inactivation of the tricarboxylate carrier of liver mitochondria by (−)-erythrofluorocitrate

Cited by 24 publications

References 7 publications

Toxicology of fluoroacetate: a review, with possible directions for therapy research

Toxicology of fluoroacetate: a review, with possible directions for therapy research

The crystal structure of rubidium ammonium hydrogen fluorocitrate dihydrate

Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate

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