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 beta-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; beta-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. beta-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 beta-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 beta-hydroxybutyrate did not. Most substrates competed between themselves, although citrate accumulation was somewhat increased by oxoglutarate. beta-Hydroxybutyrate competed for accumulation only with succinate, and was unaffected by other substrates. beta-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 K(m) values about 1-10mm. 4. It is concluded that beta-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 K(i)) 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.

Acetates↗

PubMed1969

Calcium ion-induced uptakes and transormations of substrates in liver mitochondria.

Substrate-depleted rat liver mitochondria will reaccumulate malate, succinate, oxoglutarate, beta-hydroxybutyrate and glutamate if provided with an energy source and Ca(2+) (or Ca(2+) and Mn(2+)). The energy requirement for ion uptake by fresh mitochondria causes a transient oxidation of their NADH and presumably this leads to an increased oxaloacetate concentration. A consequence is the promotion of formation of citrate, which tends to remain in the particles, provided the pH is above 7. Analyses made of systems blocked with fluorocitrate show that citrate accumulates when Ca(2+) is added with the following substrates; (a) pyruvate in the presence of ATP or malate, (b) palmitoyl-l(-)-carnitine in presence of malate and (c) oxoglutarate. Lowering the pH, even to 6.8, causes the citrate to emerge. This could be the basis of a cellular control mechanism. The generation of citrate in response to Ca(2+) can explain the stoichiometry of one proton ejected per Ca(2+) ion taken up. The new carboxyl group formed from acetyl-CoA when it condenses with oxaloacetate provides an internal anionic charge and a proton to emerge when Ca(2+) enters.

Adenosine Triphosphate↗

PubMed1969

Cation uptake as the basis for production of proton pulses by mitochondria at the anaerobic-aerobic transition.

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Acetates↗

PubMed1968

Changes of total water and sucrose space accompanying induced ion uptake or phosphate swelling of rat liver mitochondria.

1. Total water exchangeable with tritiated water and sucrose space were measured in rat liver mitochondria during the uptake of K(+) induced by valinomycin and the release caused by nigericin. The K(+) content and the sucrose-inaccessible water rose and fell together. 2. Swelling resulting from phosphate addition in a medium of high K(+) concentration was associated mainly with increased sucrose-accessible water, which carried dissolved K(+). This change was reversed by addition of ATP. 3. The response of the sucrose-inaccessible space to changed osmolarity was qualitatively that expected if the mitochondrial K(+) is assumed to be present in this space with a univalent anion. 4. It is brought out that the light-scattering method fails to distinguish between changes in sucrose space and in sucrose-inaccessible space, which in the present experiments could be altered respectively by phosphate (in high K(+) solution) and by cation uptake induced by antibiotic.

Acetates↗

PubMed1968

Effect of denervation on the synthesis of ribonucleic acid and deoxyribonucleic acid in rat diaphragm muscle.

1. The effect of unilateral denervation of rat diaphragm muscle on its content of nucleic acids and their incorporation of precursors was investigated. 2. After denervation the paralysed hemidiaphragm hypertrophies and within 3 days its content of RNA increases considerably. The concentration of DNA/unit mass remains fairly constant. 3. During this period there is some increase in the rate of incorporation of [(14)C]adenine into RNA, whereas there is some diminution in the rate of incorporation of [(14)C]orotic acid. 4. Incorporation of [(14)C]adenine and [(3)H]thymidine into DNA is much increased in the paralysed tissue, reaching its maximum by about the third day, but returning to normal by the tenth. 5. The significance of these results in relation to the hypertrophy after denervation is discussed.

Adenine↗

PubMed1968

Intramitochondrial substrate concentration as a factor controlling metabolism. The role of interanion competition.

1. The concentration-dependence of the intramitochondrial accumulation of l-malate and succinate was measured and expressed in the form of adsorption isotherms. The accumulation, however, may arise because of an internal positive potential. 2. The competition for accumulation offered by some other anions, including phosphate, was measured and is expressed conventionally by additional terms in the adsorption equation. 3. The interactions between anions were also studied when one was acting as oxidized substrate. 4. In some examples there is a parallel between the effects of an added anion on both accumulation and oxidation; in other cases chemical participation of the added substance in metabolism is presumed to remove the correlation. 5. It is suggested that by combining kinetic data on penetration with stoicheiometric data on accumulation and specific reaction rates it may be possible to account for the rates of respiration obtained with intact mitochondria. 6. It is possible to show that there is a certain phosphate/substrate ratio for maximum phosphorylation rate with some substrates. This is to be expected when phosphate and substrate compete for accumulation.

Adsorption↗