Molecular Pharmacology, Vol 14, 172-184, Copyright © 1978 by the American Society for Pharmacology and Experimental Therapeutics

Molecular Toxicology of (-)-erythro-Fluorocitrate: Selective Inhibition of Citrate Transport in Mitochondria and the Binding of Fluorocitrate to Mitochondria1 Proteins

¹ Departments of Pharmacology, Biochemistry and Biophysics, and Cardiovascular Research Institute, University of California, San Francisco, California 94143

The rate of entry of oxidizable carboxylic acids into the mitoplast compartment of lysosome-and microsome-free mitochondria was determined by monitoring substrate-dependent incorporation of ³²P-labeled orthophosphate into ADP to form ATP. The maximal rate of ATP synthesis that was dependent on tricarboxylic acids required the presence of (-)-erythro-fluoromalate, replacing the physiological activator L-malate. Previous incubation of mitochondria for 5-15 min (at 25-30°) with 50 pmoles of (-)-erythro-fluorocitrate per milligram of mitochondrial protein irreversibly and selectively inhibited citrate-supported ATP synthesis. The efflux of citrate from the mitoplast compartment of mitochondria was measured by incorporation of the acetyl residue of intramitochondrially generated citrate into fatty acids synthesized by the cytoplasmic enzyme system. Previous incubation of mitochondria for 15 min with quantities of (-)-erythro-fluorocitrate that inhibited citrate-dependent ATP synthesis also inhibited mitochondrial citrate-dependent fatty acid synthesis. The utilization of added citrate for fatty acid biosynthesis by the cytoplasmic system was not affected by fluorocitrate. Incubation of inner membrane vesicles with 3,4,5,6-¹⁴C-labeled (-)-erythro-fluorocitrate resulted in the covalent binding of fluorocitrate to protein components of the membrane as determined by molecular filtration in 8 M guanidine hydrochloride and precipitation by 10% trichloracetic acid. The fluorocitrate-containing protein precipitate could be redissolved in 0.1 N NaOH at 0° and reprecipitated by trichloracetic acid. The bond between fluorocitric acid and protein was specifically cleaved by incubation with 0.4 M neutral hydroxylamine, but not by 1 mM o-phenanthroline. The hydroxamic acid of fluorocitrate, which was formed by treatment of protein-bound fluorocitrate with hydroxylamine, was detected by high-voltage electrophoresis and thin-layer chromatography. Binding of fluorocitrate to inner membrane vesicles dissolved in 4 M guanidine hydrochloride was prevented by previous incubation of membrane proteins with the organomercurial o-{[3-(hydroxymercuri)-2-methoxypropyl]carbamoyl}phenoxyacetic acid sodium salt. The covalent binding of (—)-erythro-fluorocitrate to membrane proteins was the result of an enzymatic process that was activated by Mn²⁺. Oxidizable carboxylic acids did not form covalently bound adducts with inner membrane proteins. It was concluded that (—)-erythro-fluorocitrate specifically inhibited citrate transport by its covalent binding to two protein fractions associated with the mitoplast of liver, kidney, heart, and brain tissue.