1. The respiration of rat liver mitochondria was compared with different substrates, and with sucrose and saline media. The maximum rates of oxidation obtainable from glutamate, oxoglutarate, glutamate+malate, or succinate were higher in the saline (120mm)–tris (20mm) media than in sucrose (250mm)–tris (20mm) mixtures, but the rate with β-hydroxybutyrate was unchanged. Addition of valinomycin to a medium with sucrose and 5mm-potassium chloride led to rates similar to those measured in saline media; β-hydroxybutyrate oxidation was unaffected. 2. Some pairs of substrates together provided a rate of oxidation greater than the sum of the separate rates. This is accountable if removal of inhibitory products, such as oxaloacetate, compensates for any mutual competition between the substrates. Other pairs showed rates less than the sum of the separate rates, which is accountable by mutual competition. β-Hydroxybutyrate and other substrates, except succinate, provided strictly additive rates; with succinate there was evidence for competition. In the presence of rotenone, succinate oxidation was slowed down by citrate, oxoglutarate (+arsenite) and by β-hydroxybutyrate. 3. The accumulation of substrates in the mitochondria was measured as a function of the concentration and in the presence of possible competitors, or with a potassium salt and valinomycin to induce uptake of K+. The quantities of oxoglutarate, glutamate and pyruvate increased with the mitochondrial K+, but the quantities of β-hydroxybutyrate did not. Most substrates competed between themselves, although citrate accumulation was somewhat increased by oxoglutarate. β-Hydroxybutyrate competed for accumulation only with succinate, and was unaffected by other substrates. β-Hydroxybutyrate accumulation was almost linearly related to applied concentration (up to 5mm), and its rate of reaction was linearly dependent on concentration up to the highest value tested (0·75mm). Hence it differed from other substrates, which are accumulated and oxidized in a manner that follows a saturation law, with Km values about 1–10mm. 4. It is concluded that β-hydroxybutyrate is stored in a compartment operationally distinct from the space containing K+ and the NAD-linked substrates. It seems likely that succinate enters both compartments. 5. The degree of accumulation and the effectiveness of an anion as a competitor (as judged by low Ki) increases with the net charge. This is indicative of an electrostatic interaction with positive sites. It is suggested that the facilitating influence of dicarboxylic acids on the permeation of tricarboxylic acids may be due to the assembling of pairs of the positive carriers by the former, so favouring the chance of there being three or more carriers in a small volume of space near the boundary to interact with the tricarboxylic anion.
The dependence on dicarboxylic acids and energy of citrate accumulation in depleted rat liver mitochondria