Abstract
Rat kidney mitochondria, in the presence of ATP, Pi, and magnesium ions, converted pyruvate and bicarbonate to malate, fumarate, and citrate and, in the presence of glutamate, also to aspartate. The addition of fluorocitrate strongly inhibited pyruvate utilization and 14CO2 incorporation into organic acids. The inhibition of 14CO2 incorporation into organic acids in the presence of fluorocitrate appeared to be caused by malonyl coenzyme A produced by fluorocitrate stimulation of acetyl-CoA carboxylase. This inhibition by fluorocitrate of 14CO2 incorporation was reversed by carnitine, acetylcarnitine, acylcarnitines, isocitrate, α-ketoglutarate, succinate, fumarate, and glutamate, but not by citrate, β-hydroxybutyrate, or caprylate. The ratio of carnitine to fluorocitrate determined the degree of reversal of this inhibition. The reversal of inhibition by carnitine or acylcarnitine resulted in greatly increased citrate formation, with nearly all of the 14CO2 being incorporated into citrate, and in nearly complete inhibition of malate synthesis. Fluorocitrate inhibited oxidation of citrate. In the presence of tricarboxylic cycle intermediates, the incorporation of bicarbonate-14C into malate was greatly increased. With fluorocitrate in the system, the incorporation of radioactive bicarbonate into malate in the presence of α-ketoglutarate and succinate did not change, but decreased in the presence of fumarate.
Highlights
The once washed kidney mitochondria were suspended in 0.25 M sucrose at concentrations corresponding to 0.7 to 1.0 g of original wet tissue per ml
Mitochondria from 0.5 g of kidney were added to each incubation and contained 1.0 mg of nitrogen for Experiment 1 and 1.2 mg of nitrogen for Experiment 2
This inhibition by fluorocitrate was reversed by carnitine (Table I, Experiment 2)
Summary
Experiments were performed with intact kidney isolated according to the method of Schneider that the mitochondria were centrifuged at 15,000. CM -PRODUCTS adding perchloric acid (final concentration, 0.33 M). Acetoacetate, pyruvate, malate, a-ketoglutarate, isocitrate, glutamate, aspartate, and citrate were determined as previously described [6]. Dowex 2-formate treatment of the samples was found unnecessary [6]. Direct application of these samples on the paper strips obviated reanalysis for the organic acids after. NaHW03 was obtained from Volk and was diluted with unlabeled KHCOa to give a final concentration of about 0.2 to 0.5 PC! 250 to 399 g, from Badger Research Corporation or from Sprague-Dawley, were used in all experiments
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