Proc. Natl. Acad. Sci. USA Vol. 74, No. 11, pp. 4942-4946, November 1977 Biochemistry Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate (glutathione-S-citryl ester/metalloprotein/inner mitochondrial membrane/fluorocitrate toxic mechanism) ERNEST KUN, EVA KIRSTEN, AND MANOHAR L. SHARMA Departments of Pharmacology, Biochemistry, and Biophysics and the Cardiovascular Research Institute, University of California, San Francisco, Ca ifornia 94143 Communicated by Van R. Potter, August 29, 1977 ABSTRACT A soluble extract of the mitochondrial com- see ref. 11). The inability to demonstrate an inhibition of citrate partment composed of the inner membrane and matrix catalyzes efflux by fluorocitrate in mitochondria that had been loaded the enzymatic synthesis and hydrolysis of the 1:1 adduct of citric acid and glutathione. The adduct was identified as the thioester with citrate (9) was also explained by the stringent requirement by isolation with single and double isotope labeling ([14C]citric of preincubation of mitochondria with fluorocitrate in the ab- acid and [35S]glutathione) and by conversion to the monohy- sence of millimolar concentrations of citrate (cf. 7, 8), allowing droxamate of citric acid and comparison with the synthetic for the formation of two protein-fluorocitrate thioesters (8). product by thin layer chromatography and high voltage elec- The present report is concerned with the identification of a trophoresis. The enzymatic formation of the thioester (pH op- mitochondrial enzyme system that catalyzes the formation of timum 7.39 at 300) requires oxidized glutathione and citrate; both substrates exhibit a Michaelis-Menten kinetics. During the 1:1 adduct of glutathione and citric acid. The adduct was the enzymatic reaction equimolar quantities of thioester and identified as the thioester of glutathione, and kinetic evidence glutathione sulfinic acid are formed. After gel filtration or salt was also obtained that the mitochondrial extract contained, in fractionation the enzyme system requires Mn2+ (or Mg2+, which addition to the thioester-forming enzyme system, a hydrolase is less effective) for maximal activity. When extracts ofmitoplast capable of degrading the thioester. The citryl-glutathione- are tested, the time course of reaction is biphasic due to the forming enzyme system proved to be the most sensitive catalytic rapid synthesis of the product by the thioester-forming system (molecular weight 171,000) followed by its decay by the hydro- component of mitochondria towards the irreversible inhibitory lase (molecular weight 71,000). The two systems were separated action of (-)erythrofluorocitrate, suggesting that the molecular by molecular filtration on Sephadex G-200 and by precipitation site of action of this highly toxic agent has been identified. with (NH4)2SO4. The thioester-forming system is inhibited by preincubation with 0.5 mM mersalyl. Other inhibitors are METHODS 1,2,3propane tricarboxylic acid, 10 mM Ca2+, 200 mM K+, and the free radical trapping agent, phenazine methosulfate. The Lysosome-free mitochondria and mitoplasts (defined as the citrate-glutathione thioester formation is irreversibly and spe- matrix surrounded by the inner membrane) were isolated from cifically inhibited by (-)erythrofluorocitrate (50% inhibition rat livers as described (12). An extract of mitoplasts was pre- at 25 pmol of added fluorocitrate per mg of protein), which pared by stirring (at 00) of a suspension of mitoplasts (30 mg forms a trichloroacetic acid-stable adduct with the enzyme of protein per in M protein (at 50% inhibition, 0.8 pmol is bound to 1 mg of protein). ml) 0.25 sucrose/mannitol medium (12) Synthesis of malyl-glutathione thioester by inner membrane containing 1 mg of Brij 56 (Sigma Chemicals) per 10 mg of vesicles is selectively inhibited by (-)erythrofluoromalate. mitoplast protein for 15 min. The resulting lysate was diluted 1:3 with the medium; after removal of large granules (unbroken We have previously identified the biologically active species mitoplasts) by centrifugation at 10,000 X g (10 min at 40), the of the four possible isomers of monofluorocitric acid as supernatant (containing soluble extract of the inner membrane (-)erythrofluorocitrate (1, 2); its structure subsequently was and soluble proteins of the matrix and inner membrane vesicles) confirmed by x-ray crystallography (3, 4). Kinetic evidence was subjected to ultracentrifugation (145,000 X g for 1 hr at 20). indicated that (-)erythrofluorocitrate, when preincubated with The clear supernatant was concentrated by centrifugation (at isolated mitochondria at concentrations of 20-50 pmol of 20) in Amicon CF-25 membrane cones at 100 X g to one-fourth fluorocitrate per mg of mitochondrial protein, irreversibly in- of the original volume. Approximately 250 mg of protein ex- hibited bidirectional citrate transport (5-8). Whereas the high tract was obtained by this technique from 500 mg (protein) of sensitivity of citrate influx to low concentrations of fluorocitrate mitoplast starting material. The concentrated extract was used in intact mitochondria has been confirmed (9), an unusually directly in most experiments, unless specified, whereas the low competitive ki value for fluorocitrate was also reported for sediment containing the inner membrane vesicles was reho- the aconitase activity of disrupted mitochondria that had been mogenized in the suspending medium. Both fractions were preincubated with 1 mM Mg2+ (9). However, we have shown stored at -15° until used. The enzymatic activity of the soluble subsequently that mitochondrial aconitase is inactivated by extract decayed slowly at -150 (about half of the activity was Mg2+ in the presence of low concentrations of fluorocitrate (10) lost in 2 weeks) but much more rapidly after repeated thawing and apparent ki values calculated under these conditions have and refreezing. The activity was completely recovered when no bearing on the linearly competitive and reversible inhibition the stored extract was preincubated for 5-10 min with 5 mM of purified aconitase by (-)erythrofluorocitrate (ki = 290,M, reduced glutathione (GSH), demonstrating the essential role The costs of publication of this article were defrayed in part by the Abbreviations: GSH, reduced glutathione; GSSG, oxidized glutathione; payment of page charges. This article must therefore be hereby marked Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate citryl-SG and malyl-SG, glutathione thioesters of citric and malic acids, this fact. respectively. 4942 Downloaded by guest on October 2, 2021 Biochemistry: Kun et al. Proc. Natl. Acad. Sci. USA 74 (1977) 4943 @ - 1000 (2400 cpm) E E 0. GSH 500 un U5 A Ia' GSSG A (1, - A 'A nAnA'A Qa 0 0 0 10 20 30 40 Number of strips (1 cm each) FIG. 1. Electrophoretic isolation of the citric acid-glutathione adduct containing either 14C or 35S as radioactive label. Unreacted citric acid was removed in the preceding step (Dowex 50-H+). Citric acid placed on the paper migrated quantitatively into the buffer reservoir at the anode at pH 3.5 in 150 min. 0, 14C; A, 35S. of protein-bound -SH groups for catalytic activity. As shown GSH, 15 mM GSSG, 220,uM citrate, Tris/N-2-hydroxyethyl- later, GSH was not a substrate of the enzyme, but served as a piperazine-N'-2-ethanesulfonic acid (Hepes) buffer (10 mM, thiol-protecting agent. pH 7.4), and sucrose mannitol (0.25 M) in a final volume of 600 The glutathione-citrate adduct was quantitatively assayed ,ul. After incubation for 5 min at 300, the reaction was stopped by its isolation as follows. After incubation of the enzyme with with 2 ml of 10% trichloroacetic acid. The product was ad- oxidized glutathione (GSSG), GSH, and citrate (for details see sorbed to Dowex 50 H+ (see Methods) from six 100-,l batches Results) containing [1,5-'4C]citric acid (98 mCi/mmol; New of the incubation mixture and eluted five times with 1 M pyr- England Nuclear) in a final volume of 100 pl, the reaction was idine (2 ml for each batch). The pyridine eluates were collected stopped with 300 yl of 10% trichloroacetic acid and the pre- into one-half volume of 2 M acetic acid (at 00) in order to avoid cipitated proteins were removed by centrifugation. An aliquot alkali-catalyzed rearrangement of the thioester (16) and of the trichloroacetic acid supernatant (300 .l) was directly freeze-dried, and the product was isolated by high voltage transferred onto a small column (1 X 2 cm, containing 1.5 ml electrophoresis at pH 3.5 (0.2 M acetate/formate) and 4000 V of resin bed) of Dowex 50 H+ equilibrated with 1 M formic for 150 min at 200 (14). In the experiment shown in Fig. 1 either acid/Na formate of pH 2.0 and the unreacted citric acid was citrate, 14C, or glutathione, 35S, was labeled. Both labels ap- quantitatively removed by 12 ml of formate (1 M, pH 2.0), as peared in the same radioactive peak, corresponding to a 1:1 monitored by radiochemical analysis. After the residual formate molar ratio of citric acid and GSH. From the above large-scale was eluted from the column with 3 ml of H20, the adduct was preparative experiment 200 nmol of citrate glutathione (1:1) quantitatively eluted with 6 ml of 1 M NaOH and an aliquot adduct was obtained, which was used in part for conversion to (1.5 ml), after neutralization with 1 M HCI, was analyzed by the hydroxamate (see Methods). Comparison of the NH2OH scintillation spectrometry with toluene/Triton X-100/Omni- derivative of the enzymatically synthesized product and the fluor (1 liter:1 liter:18 g) scintillator. Labeled GSH (15S, 19 hydroxamate prepared from the chemically synthesized mCi/mmol, obtained from Schwartz/Mann) was used as the monomethylester of citric acid showed that the two products second label for the product. Hydroxamate derivatives were migrated with identical mobility, RF of 0.27, whereas RF of the prepared from synthetic monomethyl ester of citric acid (pre- unreacted citric acid was 0.18 and the RF of the 1:1 adduct was pared by the diazomethane procedure, with 0.9 mol of diazo- 0.36.