E.Jack Davis

Control of mitochondrial metabolism by the ATPADP ratio

Abstract Liver mitochondria were incubated in the presence of excess substrate, 4 mM ATP and varying concentrations of inorganic phosphate (Pi). ATPase was added to obtain steady-state rates of respiration and ATP ADPX ratios. The rates of ATPase stimulated respiration were highly correlated with control by the latter, so long as [Pi] was not limiting for phosphorylation, whereas there was strong negative correlation that the quantity [ATP] [ADP] [Pi], limited respiration either as a linear or logarithmic function. The data are consistent with the mitochondrial adenine nucleotide translocase being the rate determining reaction for respiration so long as the free ATP ADP and [Pi] are in the range which probably exist in the intact cell.

On the relationships between the stoichiometry of oxidative phosphorylation and the phosphorylation potential of rat liver mitochondria as functions of respiratory state

The characteristics of the mitochondrial adenine nucleotide exchange carrier have been studied in detail (see e.g. ref. [ 11). Exchange of ADP and ATP across the mitochondrial inner membrane, mediated by this carrier, appears to be the rate-limiting step for oxidative phosphorylation under conditions which obtain in vivo [l-3] .The asymmetry in the specificity of the transport of adenine nucleotide results in an asymmetric distribution of ADP and ATP on the two sides of the membrane, the ATP/ADP ratio being greater outside the mitochondria than inside [ 1,4, 51. As predicted by Klingenberg and co-workers [4] the formation and maintenance of this gradient of phosphorylation potential (ATP/ADP X Pi) would be expected, on thermodynamic grounds, to consume metabolic energy. The present communication reports the results of experiments carried out with liver mitochondria under conditions in which respiration is controlled by a constant and limiting supply of ADP, in such a manner that in vivo conditions are closely simulated: i.e., (a) a high extramitochondrial phosphorylation potential is maintained, and is constant throughout an incubation; and (b) the concentrations of inorganic phosphate and of extramitochondrial adenine nucleotides are held constant and within the physiological range of concentration. The data are interpreted as being a direct estimation of energy requirement for maintenance of a high cytosolic phosphorylation potential.

Regulation of glycolytic flux in an energetically controlled cell-free system: The effects of adenine nucleotide ratios, inorganic phosphate, pH, and citrate

A soluble extract from rat skeletal muscles has been used with purified mitochondrial ATPase (F1) to develop steady states with respect to glycolytic flux, the concentrations of glycolytic intermediates and inorganic phosphate, and the concentrations and ratios of adenine nucleotides. Incubations were carried out in media resembling the ionic composition in the cell cytoplasm, in an attempt to evaluate the quantitative contributions of various effectors to the overall control mechanism under simulated in vivo conditions. The primary control reaction of glycolytic flux under the conditions studied could be identified with phosphofructokinase, followed by secondary control of the reaction catalyzed by hexokinase. Glycolytic flux was increased with increasing pH over the range 6.6–7.6, both in the absence and presence of ATPase. Without other added effectors, the glycolyzing extract maintained an ATP/ADP ratio of about 50 in the pH range 7.0–7.6, and phosphofructokinase was incompletely suppressed. Addition of increasing amounts of ATPase markedly stimulated glycolytic flux coincident with lowered steady-state ATP/ADP ratios, and decreased accumulation of hexose monophosphates. Control of flux by the ATP/ADP ratio (and simultaneously altered AMP concentration) was less effective if pH (7.3 to 7.6) or phosphate concentration (2 to 20 mm) was increased. Flux through phosphofructokinase was controlled principally when the ATP/ADP ratios were varied in the range between > 50 and 15. The inhibitory effect of citrate was evaluated. Suppression of glycolytic flux and accumulation of hexose monophosphates were dependent on incubation conditions. If the pH was 7.3 or less, and the phosphate concentration low (2 mm), flux through phosphofructokinase was significantly suppressed even at citrate concentrations less than 50 μm. Simultaneous decrease in the steady-state ATP/ADP ratio and elevation of AMP was ineffective in reversing this inhibition. At higher pH and, more dramatically, when the phosphate concentration was increased, sensitivity to citrate inhibition was markedly diminished. These data, taken together with studies of respiratory control with isolated mitochondria (E. J. Davis, and L. Lumeng, 1975), J. Biol. Chem.250, 2275–2282) strongly suggest that adenine nucleotide control of both glycolysis and respiration is exerted when the ratio of free nucleotides (not protein bound) in the cytosol is in the range of 15 to > 50. The data further suggest that citrate plays an important role in the regulation of glycolysis in muscle when the ATP/ADP ratio is high (and the phosphate concentration is correspondingly low), but that this inhibition is overcome by liberation of inorganic phosphate during muscle contraction.

The effect of acylcarnitines on the oxidation of branched chain α-keto acids in mitochondria

Abstract The oxidation of 14 C-labelled branched-chain α-keto acids corresponding to the branched-chain amino acids valine, isoleucine and leucine has been studied in isolated mitochondria from heart, liver and skeletal muscle. 1. 1. Heart and liver mitochondria have similar capacities to oxidize these α-keto acids based on protein content. Skeletal muscle mitochondria also show significant activity. 2. 2. Half maximum rates are obtained with approximately 0.1 mM of the α-keto acids under optimal conditions. Added NAD and CoA had no effect on the oxidation rate, showing that endogenous mitochondrial NAD and CoA are required for the oxidation. 3. 3. Addition of camitine esters of fatty acids (C 6 –C 16 ), succinate, pyruvate, or α-ketoglutarate inhibited the oxidation of the branched chain α-keto acids, especially in a high-energy state (no ADP added). In heart mitochondria the addition of ADP (low-energy state) decreased the inhibitory effects of acylcarnitines of medium chain length or of pyruvate, and abolished the inhibitory effect of succinate. It is suggested that the oxidation rate is regulated mainly by the redox state of the mitochondria under the conditions used. 4. 4. The results are discussed in relation to the regulation of branched-chain amino acid metabolism in the body.

Studies on the active transfer of reducing equivalents into mitochondria via the malate-aspartate shuttle

1. The effects of mitochondrial energy states onthe extramitochondrial NADH/NAD ratio via a reconstituted malate-aspartate shuttle have been investigated. 2. The transfer of reducing equivalents into isolated mitochondria is stimulated by ATP and by electron transport. The effect of ATP is inhibited by oligomycin. The effect of electron transport is inhibited by uncouplers. 3. Uncoupling of the mitochondria is required for rapid transfer of reducing equivalents out of the mitochondria. 4. A glutamate-stimulated entry of aspartate into energized mitochondria suggests that the malate-aspartate shuttle is to some extent reversible even in a high energy state of the mitochondria. 5. It is concluded that the malate-aspartate shuttle contributes to the formation of the skewed redox situation across the inner mitochondrial membrane, which has a more reduced inside.

On the nature of malonate-insensitive oxidation of pyruvate and glutamate by heart sarcosomes.

Abstract 1. A quantitative evaluation of respiration of isolated heart sarcosomes due to pyruvate and glutamate has been made when the citric acid cycle is blocked. 2. Malonate inhibits respiration due to added pyruvate 95–97% when phosphate and ADP are present. Pyruvate is converted quantitatively to acetoacetate and acetate under these conditions. 3. Malonate-insensitive removal of pyruvate and the appearance of acetoacetate and acetate depend on the presence of phosphate and ADP. This requirement is not prevented by 2,4-dinitrophenol, but is circumvented by arsenate. 4. When sarcosomes are incubated in the presence of 2,4-dinitrophenol and arsenite, added glutamate is slowly converted to α-oxoglutarate. Further addition of pyruvate does not stimulate the rate of α-oxoglutarate accumulation. These results are interpreted as showing the presence of glutamate dehydrogenase (EC 1.4.1.3) whereas alanine aminotransferase activity was not detectable.

The oxidation of acetate by liver mitochondria

Acetate produced from ethanol is oxidized to only a very limited extent by the liver [ 11, even though the enzymes required for activation of acetate to acetyl CoA are present in liver mitochondria in quantities adequate for a substantial rate of oxidation [ 21. A number of studies have emphasized the fact that the rate of CO? production from acetate and thus, reactions of the citric acid cycle by the liver are markedly suppressed as a result of the mitochondrial oxidation of NADH produced in the cytosol during the oxidation of ethanol [3,4]. However, these studies do not distinguish between effects on the activation of acetate from those on its further oxidation. The data presented in this communication show that the mitochondrial oxidation of acetate is limited by the rate of recycling of AMP produced during its activation. This limitation is overcome by supporting substrates which generate GTP, but is reinstated by added NADH when it competes with these substrates for oxidation. Links between substrate-linked phosphorylation and the oxidation of long-chain fatty acids by rat liver mitochondria [5], and of acetate oxidation by sheep liver mitochondria have recently been reported [6].

Carnitine palmitoyltransferase: activation and inactivation in liver mitochondria from fed, fasted, hypo- and hyperthyroid rats

The sensitivity of carnitine palmitoyltransferase to malonyl-CoA is lost when liver mitochondria are preincubated in a KCl-containing medium. This loss of sensitivity is slowed down in mitochondria from hypothyroid rats and accelerated in mitochondria from fasted and hyperthyroid rats. Glucagon seems to enhance the effect of fasting. The loss of sensitivity is significantly slowed down by 50-500 nM malonyl-CoA and accelerated by small amounts of palmitoyl-CoA in the preincubation medium.

The effect of pyrvate on cyclic oxidations by heart sarcosomes

Abstract 1. The stoichiometry of oxidation of pyruvate and glutamate by heart mitochondria has been reinvestigated. 2. The inhibition by pyruvate of its own oxidation and that of glutamate is shown to be due not to a side reaction, but to a trace amount of contaminant (probably parapyruvate) which inhibits reversibly the reoxidation of cyclicly generated α-oxoglutarate. 3. Enzymatically generated pyruvate and purified pyruvate are oxidised at a constant rate by heart mitochondria in the absence of added malate without significant accumulation of intermediates, whereas crystalline sodium pyruvate is very poorly oxidised, and causes the accumulation of α-oxoglutarate. 4. Glutamate oxidation is optimal without addition of malate. In the presence of endogenous substrates or very low concentration of malate, glutamate stimulates pyruvate disappearance. These observations are explained by a very slow net synthesis of circulating intermediates from glutamate.

Citrate as a regulator of acetyl-CoA metabolism in liver mitochondria

Abstract 1. 1.|The reversibility of citrate synthesis and the effects of citrate and fluorocitrate on citrate synthesis in whole mitochondria have been investigated. 2. 2.|Cleavage of citrate to oxaloacetate (trapped as malate) and acetyl-CoA (trapped as acetylcarnitine) in whole liver mitochondria can be demonstrated. The maximum rate of this reversed reaction is at most 1 40 of the maximum rate of citrate synthesis. 3. 3.|Citrate and fluorocitrate are competitive inhibitors with respect to oxaloacetate of purified pig heart synthase. The K i for both compounds was found to be approx. 1.5 mM. 4. 4.|Both citrate and fluorocitrate inhibit citrate synthesis and increase ketogenesis in whole liver mitochondria. They probably act as competitive inhibitors with respect to oxaloacetate, since malate counteracts this inhibition. 5. 5.|For kinetic reasons it is concluded that the citrate synthesis reaction probably never approaches equilibrium in the intact tissue. The importance of citrate in the regulation of citrate and ketone body formation in the liver is discussed.