1. Pyruvate equilibrates, like other carboxylic acids, across the membrane of rat-liver mitochondria according to the transmembrane pH difference.2. Comparison of the rate of pyruvate, acetoacetate and acetate efflux from mitochondria as well as a study of the effect of mersalyl and N-ethylmaleimide on this process, show that the diffusion of pyruvic and acetoacetic acid across the mitochondrial membrane is carrier-mediated. The process might consist of pyruvate (acetoacetate) hydroxyl ion exchange-diffusion or of pyruvic acid (acetoacetic acid) uniport. On the contrary acetic acid appears to diffuse freely across the membrane.3. The initial rate of the net pyruvate uptake by rat liver mitochondria follows saturation kinetics. The increase of the external pH of the medium as well as the addition of acetoacetate inhibit competitively pyruvate uptake. Pyruvate uptake is inhibited by mersalyl and N-ethylmaleimide.4. Mitochondria1 [14C]pyruvate exchanges with unlabelled pyruvate and acetoacetate but not with other monocarboxylates, Pi and citric-acid cycle intermediates.5. The pyruvate-pyruvate and pyruvate-acetoacetate exchanges follow saturation kinetics and exhibit the same V but different K, values. Both the exchanges are inhibited by mersalyl, but not by N-ethylmaleimide.6. It is concluded that pyruvate translocation across the mitochondrial membrane is mediated by a specific transport system. Mono-, di-and tri-carboxylic substrates appear to equilibrate across the mitochondrial membrane according to the transmembrane pH difference [l, 21. This may be attained either through free diffusion of the undissociated acids or by means of carriermediated, direct or indirect, exchange-diffusion of the anionic species with hydroxyl ions (this process is practically equivalent to anion-H f symport) [l -41
(see however [5]).The movement across the mitochondrial membrane of di-and tri-carboxylic substrates has been shown to be mediated by specific translocators [3,4,6,7]. It has, on the other hand, been proposed that monocarboxylic acids diffuse freely across the mitochondrial membrane [6]. Since undissociated carboxylic acids, but not the anionic species, are significantly lipidsoluble, it is likely that at neutral pH a free-diffusion across the membrane of weak acids takes place. Such a process might, in fact, occur at a significant rate in the case of weak monocarboxylic acids like acetate (pK, = 4.75), propionate (pK, = 4.88) and higher unsubstituted homologues, but should be negligible for relatively stronger acids like for example pyruvate which has a pK, of 2.5 [8].These considerations prompted us to examine, systematically, the transport of this substrate in isolated mitochondria, a topic which so far has received only limited attention [9,10]. Elucidation of the mechanism of pyruvate transport in mitochondria appears to be particularly desirable in the light of the fact that this substrate, produced by glycolysis in the cytosol, is the main physiological feeder of the citric-acid cycle in mitochondria.In previous r...