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. 1977 Nov;74(11):4942–4946. doi: 10.1073/pnas.74.11.4942

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

Ernest Kun 1,2,3,4, Eva Kirsten 1,2,3,4, Manohar L Sharma 1,2,3,4
PMCID: PMC432073  PMID: 270728

Abstract

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. The adduct was identified as the thioester by isolation with single and double isotope labeling ([14C]citric acid and [35S]glutathione) and by conversion to the monohydroxamate of citric acid and comparison with the synthetic product by thin layer chromatography and high voltage electrophoresis. The enzymatic formation of the thioester (pH optimum 7.39 at 30°) requires oxidized glutathione and citrate; both substrates exhibit a Michaelis-Menten kinetics. During the enzymatic reaction equimolar quantities of thioester and glutathione sulfinic acid are formed. After gel filtration or salt fractionation the enzyme system requires Mn2+ (or Mg2+, which is less effective) for maximal activity. When extracts of mitoplast are tested, the time course of reaction is biphasic due to the rapid synthesis of the product by the thioester-forming system (molecular weight 171,000) followed by its decay by the hydrolase (molecular weight 71,000). The two systems were separated by molecular filtration on Sephadex G-200 and by precipitation with (NH4)2SO4. The thioester-forming system is inhibited by preincubation with 0.5 mM mersalyl. Other inhibitors are 1,2,3-propane tricarboxylic acid, 10 mM Ca2+, 200 mM K+, and the free radical trapping agent, phenazine methosulfate. The citrate-glutathione thioester formation is irreversibly and specifically inhibited by (-)erythrofluorocitrate (50% inhibition at 25 pmol of added fluorocitrate per mg of protein), which forms a trichloroacetic acid-stable adduct with the enzyme protein (at 50% inhibition, 0.8 pmol is bound to 1 mg of protein). Synthesis of malyl-glutathione thioester by inner membrane vesicles is selectively inhibited by (-)erythrofluoromalate.

Keywords: glutathione-S-citryl ester, metalloprotein, inner mitochondrial membrane, fluorocitrate toxic mechanism

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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