The steady state concentrations of coenzyme A-SH and coenzyme A thioester, citrate, and isocitrate during tricarboxylate cycle oxidations in rabbit heart mitochondria.

The steady state mitochondrial content of coenzyme A-SH (CoA), acetyl-CoA, succinyl-CoA, and long chain acyl-CoA has been determined during the oxidation of palmitoylcarnitine by rabbit heart mitochondria. Variation of the substrate concentration during ADP-stimulated (state 3) respiration varies the mitochondrial content of long chain acyl-CoA and the rate of O2 uptake, and permits the conclusion that the Km of beta oxidation for intramitochondrial long chain acyl-CoA is approximately 1 nmol/mg of mitochondrial protein. At near saturating concentrations of palmitoylcarnitine, plus L-malate, the addition of ADP causes a decrease in acetyl-CoA, an increase in CoA and succinyl-CoA, and no clear change in long chain acyl-CoA content. These changes reverse upon the depletion of ADP (state 3 leads to 4 transition). Similar changes in CoA, acetyl-CoA, and succinyl-CoA are seen during state 4 leads to 3 leads to 4 transitions with pyruvate plus L-malate and octanoate plus L-malate as substrates. These results suggest a limitation of flux by citrate synthase during the controlled oxidation of these three substrates. The ratio acetyl-CoA/succinyl-CoA was determined not only during state 3 and state 4 oxidation of palmitoylcarnitine plus L-malate and pyruvate plus L-malate, but also during intermediate respiratory states (state 3 1/2) generated by adding glucose and varying amounts of hexokinase. These intermediate states are characterized by a high succinyl-CoA content, relative to either state 3 or state 4, and a suboptimal flux through citrate synthase, estimated either by pyruvate disappearance or by O2 uptake.     

Effect of micromolar concentrations of free Ca2+ ions on pyruvate dehydrogenase interconversion in intact rat heart mitochondria.

1. The mitochondrial content of active (dephospho) pyruvate dehydrogenase (PDHA) was found to be severalfold higher at an extramitochondrial Ca2+ concentration of 2 microM (pCa6) than at pCa7. The nature of the respiratory substrate did not affect this finding. 2. This Ca2+-dependence was shown in state-4 and 50%-state-3 conditions [see Chance & Williams (1956) Adv. Enzymol. 17, 65-134], but was absent in the presence of excess ADP (state 3). 3. Na+ and Mg2+ ions shifted the pCa value required for a maximal PDHA content to lower values. This was attributed to a stimulation of mitochondrial Ca2+ egress and an inhibition of uptake, respectively. Na+ ions diminished pyruvate dehydrogenase phosphate phosphatase activity in mitochondria which had been extensively depleted of Ca2+ ions by incubation with EGTA, raising the possibility of a direct inhibitory effect of Na+ ions, unrelated to Ca2+ movements. 4. Mg2+ ions lowered the mitochondrial PDHA content at pCa 6.24 and 6.48, but had only minimal effects in the presence of EGTA. 5. The effects of P1 and bicarbonate ions on PDHA content were also studied, as possible effectors of mitochondrial Ca2+ transport. Bicarbonate ions abolished the response to Ca2+ ions, by generating maximal values of PDHA content, but such a response was still observed when physiological concentrations of both P1 and bicarbonate were used. 6. The pCa of the medium in the range 6.33 to over 7 affected PDHA content, with only very minor changes in state-4 rates of O2 uptake and no change in [ATP]/[ADP] ratio or in mitochondrial [NADH]/[NAD+] ratio, provided that Mg2+ ions were present. Thus the effect of Ca2+ ions on PDHA content is unlikely to be mediated by changes in [ATP]/[ADP] and [NADH]/[NAD+] ratio and is more likely to be direct. Equally, changes in the [acetyl-CoA]/[CoA] ratio in response to Ca2+ ions when the substrate was pyruvate were the converse of those required to mediate changes in interconversion, and are probably secondary to changes in PDHA content.     

Metabolism and transport of glutamine and glucose in vascularly perfused small intestine of the rat

1. The proportion of active (dephosphorylated) pyruvate dehydrogenase in rat heart mitochondria was correlated with total concentration ratios of ATP/ADP, NADH/NAD+ and acetyl-CoA/CoA. These metabolites were measured with ATP-dependent and NADH-dependent luciferases. 2. Increase in the concentration ratio of NADH/NAD+ at constant [ATP]/[ADP] and [acetyl-CoA]/[CoA] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between mitochondria incubated with 0.4mM- or 1mM-succinate and mitochondria incubated with 0.4mM-succinate+/-rotenone. 3. Increase in the concentration ratio acetyl-CoA/CoA at constant [ATP]/[ADP] and [NADH][NAD+] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between incubations in 50 micrometer-palmitotoyl-L-carnitine and in 250 micrometer-2-oxoglutarate +50 micrometer-L-malate. 4. These findings are consistent with activation of the pyruvate dehydrogenase kinase reaction by high ratios of [NADH]/[NAD+] and of [acetyl-CoA]/[CoA]. 5. Comparison between mitochondria from hearts of diabetic and non-diabetic rats shows that phosphorylation and inactivation of pyruvate dehydrogenase is enhanced in alloxan-diabetes by some factor other than concentration ratios of ATP/ADP, NADH/NAD+ or acetyl-CoA/CoA.     

Inhibition of lipogenesis by halothane in isolated rat liver cells.

1. Halothane at clinically effective concentrations [2.5 and 4% (v/v) of the gas phase of the incubation flask] was found to inhibit significantly lipogenesis from endogenous substrates, e.g., glycogen, or from added lactate plus pyruvate. This was accompanied by a decrease in the ratio of the free [NAD+]/[NADH] of the mitochondrion and the cytoplasm, as shown by the [3-hydroxybutyrate]/[acetoacetate] ratio and the [lactate]/[pyruvate] ratio. 2. Acetoacetate or pyruvate decreased the inhibitory effect of halothane and restored lipogenesis to control rates. They were reduced rapidly by 3-hydroxybutyrate dehydrogenase or lactate dehydrogenase respectively, with the concomitant oxidation of NADH and the generation of NAD+. 3. These results suggest that the mechanism by which halothane inhibits lipogenesis from glycogen or lactate is by inhibition of the oxidation of NADH; this results in inhibition of flux of carbon through pyruvate dehydrogenase and a shortage of acetyl-CoA for fatty acid synthesis. Thus when NADH acceptors are added in the presence of halothane, the concentration of mitochondrial NAD+ is raised so that the flux of carbon through pyruvate dehydrogenase increases and lipogenesis is restored.     

Regulation of fatty acid beta-oxidation in rat heart mitochondria

In an attempt to elucidate the mechanism by which the rate of fatty acid oxidation is tuned to the energy demand of the heart, the effects of changing intramitochondrial ratios of [acetyl-CoA]/[CoASH] and [NADH]/[NAD+] on the rate of beta-oxidation were studied. When 10 mM L-carnitine was added to coupled rat heart mitochondria to lower the ratio of [acetyl-CoA]/[CoASH], the rate of palmitoylcarnitine beta-oxidation, as measured by the formation of acid-soluble products, was stimulated more than fourfold at state 4 respiration while beta-oxidation at state 3 respiration was hardly affected. Neither oxaloacetate nor acetoacetate, added to mitochondria to lower the [NADH]/[NAD+] ratio, stimulated beta-oxidation. Rates of respiration at states 3 and 4 were unchanged by additions of L-carnitine, oxaloacetate, or acetoacetate. Determinations of intramitochondrial ratios of [acetyl-CoA]/[CoASH] by high performance liquid chromatography yielded values close to 10 for palmitoylcarnitine-supported respiration at state 4 and 2.5 at state 3 respiration. Addition of 10 mM L-carnitine caused a dramatic decrease of these ratios to less than 0.2 at both respiration states. Studies with purified or partially purified enzymes revealed strong inhibitions of 3-ketoacyl-CoA thiolase by acetyl-CoA and of L-3-hydroxyacyl-CoA dehydrogenase by NADH. Moreover, the activity of 3-ketoacyl-CoA thiolase at concentrations of acetyl-CoA and CoASH prevailing at state 3 respiration was 4 times higher than its activity in the presence of acetyl-CoA and CoASH observed at state 4. Altogether, this study leads to the conclusion that the rate of beta-oxidation in heart can be regulated by the intramitochondrial ratio of [acetyl-CoA]/[CoASH] which reflects the energy demand of the tissue. The thiolytic cleavage catalyzed by 3-ketoacyl-CoA thiolase may be the site at which beta-oxidation is controlled by the [acetyl-CoA]/[CoASH] ratio.     

The redox state of the nicotinamide–adenine dinucleotides in rat liver homogenates

1. The redox state of the NAD couple of rat liver mitochondria, as measured by the [beta-hydroxybutyrate]/[acetoacetate] ratio, rapidly changed in the direction of oxidation during the preparation of homogenates in a saline medium. The value of the [beta-hydroxybutyrate]/[acetoacetate] ratio fell from 2.3 to 0.15 in 10min. EDTA diminished the fall and succinate prevented it. 2. The redox state of the rat liver cytoplasm, as measured by the [lactate]/[pyruvate] ratio, changed slightly in the direction of reduction during the preparation of homogenate. This was prevented by succinate. 3. In unsupplemented homogenates the differences in the redox states of mitochondria and cytoplasm decreased. Succinate and EDTA together maintained the differences within the physiological range. A measure of the ability of the mitochondria to maintain different redox states in mitochondria and cytoplasm is the value of the expression [lactate][acetoacetate]/[pyruvate][beta-hydroxybutyrate]. If there are no differences in the redox states of the NAD in the two cell compartments the value of the expression is 444 at 37 degrees . The value in the intact rat liver is between 4.7 and 21. 4. alpha-Oxoglutarate or glutamate were still more effective than succinate in maintaining high [beta-hydroxybutyrate]/[acetoacetate] ratios in the homogenates because these substrates supply a reducing agent of NAD(+) and, through succinate, an inhibitor of the oxidation of NADH. 5. When supplemented with alpha-oxoglutarate and EDTA, homogenates readily adjust the redox state of the beta-hydroxybutyrate dehydrogenase system after it has been upset by the addition of either acetoacetate or beta-hydroxybutyrate. 6. Amytal and rotenone raised the value of the [beta-hydroxybutyrate]/[acetoacetate] ratio. This is taken to indicate that the reduction of acetoacetate in the homogenates was not an energy-linked process. 7. 2,4-Dinitrophenol shifted the [beta-hydroxybutyrate]/[acetoacetate] ratio in the presence of succinate in favour of oxidation because it inhibited the oxidation of succinate and accelerated the oxidation of NADH. 8. Rotenone increased the rate of ketone-body formation of liver homogenates, though it decreased the rate of oxygen uptake.     

Effect of carnitine on mitochondrial oxidation of palmitoylcarnitine

The effects of carnitine on the metabolism of palmitoylcarnitine were studied by using isolated rat liver mitochondria. Particular attention was given to carnitine acyltransferase-mediated interactions between carnitine and the mitochondrial CoA pool. Carnitine concentrations less than 1.25mm resulted in an increased production of acetylcarnitine during palmitoylcarnitine oxidation. Despite this shunting of C₂ units to acetylcarnitine formation, no change was observed in the rate of oxygen consumption or major product formation (citrate or acetoacetate). Further, no changes were observed in the mitochondrial content of acetyl-CoA, total acid-soluble CoA or acid-insoluble acyl-CoA. These observations support the concept, based on studies in vivo, that the carnitine/acylcarnitine pool is metabolically sluggish and the acyl-group flux low as compared with the CoA/acyl-CoA pool. Acid-insoluble acyl-CoA content was decreased and CoA content increased at carnitine concentrations greater than 1.25mm. When [¹⁴C]carnitine was used in the incubations, it was demonstrated that this resulted from acid-insoluble acylcarnitine formation from intramitochondrial acid-insoluble acyl-CoA mediated by carnitine palmitoyltransferase B. Again, the higher carnitine concentrations resulted in no changes in the rates of oxygen consumption or major product formation. The above effects of carnitine were observed whether citrate or acetoacetate was the major product of oxidation. In contrast, an increase in acetyl-CoA concentration was observed at high carnitine concentrations only when acetoacetate was the product. Since the rate of acetoacetate production was not changed, these higher acetyl-CoA concentrations suggest that a new steady state had been established to maintain acetoacetate-production rates. Since there was no change in acetyl-CoA concentration when citrate was the major product, a change in the activity of the pathway utilizing acetyl-CoA for ketone-body synthesis and the potential regulation of this pathway must be considered.     

Relative importance of pyruvate dehydrogenase interconversion and feed-back inhibition in the effect of fatty acids on pyruvate oxidation by rat heart mitochondria.

Rat heart mitochondria have been incubated with concentrations of pyruvate from 50 to 500 μm as substrate in the presence or absence of an optimal concentration of palmitoylcarnitine and with respiration limited by phosphate acceptor. The rate of pyruvate utilization has been determined and compared with the amount of active (dephosphorylated) pyruvate dehydrogenase measured in samples of mitochondria taken throughout the experiments and assayed under V_max conditions. At a given pyruvate concentration, differences in pyruvate utilization as a proportion of the content of active pyruvate dehydrogenase are attributed to differences in feed-back inhibition and interpreted in terms of the ratios of mitochondrial $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ and CoA/acetyl-CoA. Under most conditions, differences in inhibition can be attributed to differences in the CoA/acetyl-CoA ratio. Feed-back inhibition is very pronounced in the presence of palmitoylcarnitine. These parameters are also examined in the presence of dichloroacetate, used to raise the steady-state levels of active pyruvate dehydrogenase in the absence of changing the pyruvate concentration, and in the presence of various ratios of carnitine/acetylcarnitine, which predominantly change the mitochondrial CoA/acetyl-CoA ratio. The latter experiment provides evidence that a decrease in mitochondrial $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio from 3.5 to 2.2 essentially balances an increase in CoA/acetyl-CoA ratio from 0.67 to 12 in modulating enzyme interconversion, whereas the change in CoA/acetyl-CoA ratio is preponderant in effecting feed-back inhibition. Increasing the free Ca²⁺ concentration of incubation media from 10^?7 to 10^?6m using CaCl₂-ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid buffers is shown to increase the steady-state level of active pyruvate dehydrogenase in intact mitochondria, in the absence of added ionophores. Moreover, this activation is reversible. Effects of Ca²⁺ ions are dependent upon the poise of the enzyme interconversion system and were only seen in these experiments in the presence of palmitoylcarnitine.     

Studies on the effects of coenzyme A-SH: acetyl coenzyme A, nicotinamide adenine dinucleotide: reduced nicotinamide adenine dinucleotide, and adenosine diphosphate: adenosine triphosphate ratios on the interconversion of active and inactive pyruvate dehydrogenase in isolated rat heart mitochondria.

The content of coenzyme A-SH (CoASH) and acetyl-CoA of suspensions of rat heart mitochondria was stabilized by the addition of DL-carnitine and acetyl-DL-carnitine, in the presence of the respiratory inhibitor rotenone. The mitochondrial content of NAD+ and NADH was similarly stabilized by the addition of acetoacetate and DL-3-hydroxybutyrate, and the content of ADP and ATP was imposed by the addition of these nucleotides to the mitochondrial suspension, in the presence of uncoupling agent and oligomycin, to inhibit ATPase. Under these conditions, mitochondrial CoASH/acetyl-CoA, NAD+/ NADH, and ADP/ATP ratios could be varied independently, and the effect on the interconversion of active and inactive pyruvate dehydrogenase could be studied. Decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios were shown to be inhibitory to the steady state activity of pyruvate dehydrogenase, and this effect is described at three different ADP/ATP ratios and different concentrations of added MgCl2. A new steady state level of activity was achieved within 10 min of a change in either CoASH/acetyl-CoA or NAD+/NADH ratio; the rate of inactivation was much higher than the rate of reactivation under these conditions. Effects of CoASH/acetyl-CoA and NAD+/NADH may be additive but are still quantitatively lesser than the changes in activity of pyruvate dehydrogenase induced by changes in ADP/ATP ratio. The variation in activity of pyruvate dehydrogenase with ADP/ATP ratio is described in the absence of changes in the other two ratios, conditions which were not met in earlier studies which employed the oxidation of different substrates to generate changes in all three ratios.     

Evidence that the flux control coefficient of the respiratory chain is high during gluconeogenesis from lactate in hepatocytes from starved rats. Implications for the hormonal control of gluconeogenesis and action of hypoglycaemic agents.

1. Increasing concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a mild respiratory-chain inhibitor [Halestrap (1987) Biochim. Biophys. Acta 927, 280-290], caused progressive inhibition of glucose production from lactate + pyruvate by hepatocytes from starved rats incubated in the presence or absence of oleate and gluconeogenic hormones. 2. No significant changes in tissue ATP content were observed, but there were concomitant decreases in ketone-body output and cytochrome c reduction and increases in NADH fluorescence and the ratios of [lactate]/[pyruvate] and [beta-hydroxybutyrate]/[acetoacetate]. 3. The inhibition by DCMU of palmitoylcarnitine oxidation by isolated liver mitochondria was used to calculate a flux control coefficient of the respiratory chain towards gluconeogenesis. In the presence of 1 mM-oleate, the calculated values were 0.61, 0.39 and 0.25 in the absence of hormone and in the presence of glucagon or phenylephrine respectively, consistent with activation of the respiratory chain in situ as previously suggested [Quinlan & Halestrap (1986) Biochem. J. 236, 789-800]. 4. Cytoplasmic oxaloacetate concentrations were shown to decrease under these conditions, implying inhibition of pyruvate carboxylase. 5. Inhibition of gluconeogenesis from fructose and dihydroxyacetone was also observed with DCMU and was accompanied by an increased output of lactate + pyruvate, suggesting that activation of pyruvate kinase was occurring. With the latter substrate, measurements of tissue ADP and ATP contents showed that DCMU caused a small fall in [ATP]/[ADP] ratio. 6. Two inhibitors of fatty acid oxidation, pent-4-enoate and 2-tetradecylglycidate, were shown to abolish and to decrease respectively the effects of hormones, but not valinomycin, on gluconeogenesis from lactate + pyruvate, without changing tissue ATP content. 7. It is concluded that the hormonal increase in mitochondrial matrix volume stimulates fatty acid oxidation and respiratory-chain activity, allowing stimulation of pyruvate carboxylation and thus gluconeogenesis to occur without major changes in [ATP]/[ADP] or [NADH]/[NAD+] ratios. 8. The high flux control coefficient of the respiratory chain towards gluconeogenesis may account for the hypoglycaemic effect of mild respiratory-chain inhibitors.     

Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide.

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.     

The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The steady-state concentrations of coenzyme A, acetyl-coenzyme A and succinyl-coenzyme A in flight muscle and isolated mitochondria

(1) A ;cycling' method involving citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) was modified by the inclusion of succinyl-CoA synthetase (EC 6.2.1.5) and hexokinase (EC 2.7.1.1) to permit the determination of very small amounts of succinyl-CoA in addition to CoA and acetyl-CoA. (2) Application of this technique to blowfly (Phormia regina) flight-muscle extracts reveals no change in acetyl-CoA concentration, a slight fall in CoA concentration and a rise in succinyl-CoA concentration during flight. (3) Extraction of isolated mitochondria during controlled (state 4) pyruvate oxidation reveals essentially only acetyl-CoA. Activation of respiration by ADP (state 3) or uncoupling agents leads to a fall in acetyl-CoA and a rise in CoA and succinyl-CoA content. (4) The presence of glycerol phosphate in addition to pyruvate results in a lower acetyl-CoA content in state 4. (5) It is contended that these results are consistent with a primary control of one of the reactions of the tricarboxylate cycle, rather than of pyruvate dehydrogenase, during the state 4 oxidation of pyruvate by isolated mitochondria, and that the modulation of citrate synthase activity by the ratio of acetyl-CoA/succinyl-CoA is unimportant under these conditions.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.     

Substrate inhibition in the tricarboxylic acid cycle

It has been shown in the experiments on rat liver mitochondria under glucose hexo-kinase load that excess of substrates of (1-20 mM) pyruvate, acetate, propionate, pent-4-enoate and malate may induce oxidation of NAD(P)H and inhibition of mitochondrial respiration (by 20-50% and more) due to a decreased rate of hydrogen production by tricarboxylic acid cycle. It has been concluded from the analysis of mathematical models and metabolite-testings which remove this inhibition that for pyruvate and acetate this inhibition is an autocatalytic one. It is related to a decreased level of CoA and oxaloacetate due to the formation of "traps" such as acetyl-CoA and alpha-kotoglutarate. For propionate and pent-4-enoate in the bicarbonate-free medium suppression of the flux in the cycle is concerned with a decreased level of CoA, acetyl-CoA and succionoyl CoA due to the accumulation of propionyl-CoA. It seems to be also concerned with the inhibition of citrate-synthetase and alpha-ketoglutarate-dehydrogenase by propionyl-CoA. Malate (in the presence of malonate) can inhibit respiration at the expense of direct inhibition of citrate-synthetase.     

Evidence for the regulatory function of synaptoplasmic acetyl-CoA in acetylcholine synthesis in nerve endings.

Isolated synaptosomes maintained a relatively stable level of acetyl-CoA during their incubation in the presence of 30 mM-KCl. Addition of Ca2+ resulted in inhibition of pyruvate oxidation and slight activation of acetylcholine synthesis. The cation decreased acetyl-CoA in intrasynaptosomal mitochondria, but did not alter its content in synaptoplasm. Verapamil did not affect pyruvate oxidation, but decreased acetyl-CoA in synaptoplasm and inhibited acetylcholine synthesis in synaptosomes. It indicates that Ca2+ might regulate acetylcholine synthesis through changes in the direct transfer of acetyl-CoA from mitochondria to synaptoplasm.     

The physiological role of oxygen-sensitive pyruvate dehydrogenase in mitochondrial fatty acid synthesis in Euglena gracilis

In Euglena gracilis a malonyl-CoA-independent fatty acid-synthetic system, in which fatty acids are synthesized directly from acetyl-CoA as both primer and C2 donor, occurs in mitochondria, and the system contributes to the wax ester fermentation. The activity of fatty acid synthesis in the mitochondrial system was enhanced about six times when an artificial acetyl-CoA-regenerating system was present, indicating that the fatty acid-synthetic activity is controlled by the ratio of acetyl-CoA against CoA. When fatty acids were synthesized using pyruvate instead of acetyl-CoA as substrate, a high activity, about 30 times higher than that from acetyl-CoA, was found under anaerobic conditions (below 10(-5) M oxygen), while in aerobiosis fatty acids were not synthesized at all. CoA, NADH, and NADP+ were required as cofactors for fatty acid synthesis from pyruvate. It was indicated that high activity of fatty acid synthesis from pyruvate due to the high ratio of acetyl-CoA against CoA was maintained by the action of the oxygen-sensitive pyruvate dehydrogenase found in Euglena mitochondria. When [2-14C]pyruvate was fed into intact mitochondria under anaerobic conditions, radioactive fatty acids were formed in the presence of malate, which provided reducing power for the matrix.     

Regulation of pyruvate oxidation in mitochondria isolated from fetal and adult rat liver

The effect of ATP on the velocity of oxygen uptake during the oxidation of pyruvate plus malate, in the presence of oligomycin, 2,4-dinitrophenol and fluorocitrate, was studied in mitochondria, isolated from the livers of adult and fetal rats.It was found that the addition of ATP caused an inhibition in the rate of oxygen uptake of 21 ± 6% in mitochondria from adult rat liver and 49 ± 8% in mitochondria from fetal rat liver. Measurements of the velocity of oxygen uptake during the oxidation of pyruvate plus malate and of palmitoylcarnitine in adult rat liver mitochondria in the presence of ATP showed that the activity of pyruvate dehydrogenase was lower than the activity of citrate synthase.In fetal mitochondria, addition of ATP resulted in an increase in the CoASH/acetyl-CoA ratio, indicating that pyruvate dehydrogenase was rate limiting here as well.It is concluded that ATP inhibited pyruvate oxidation by phosphorylation of the pyruvate dehydrogenase complex, rather than by inhibiting citrate synthase under these conditions.     

Reversible phosphorylation of pyruvate dehydrogenase in rat skeletal-muscle mitochondria. Effects of starvation and diabetes.

The total activity of pyruvate dehydrogenase (PDH) complex in rat hind-limb muscle mitochondria was 76.4 units/g of mitochondrial protein. The proportion of complex in the active form was 34% (as isolated), 8-14% (incubation with respiratory substrates) and greater than 98% (incubation without respiratory substrates). Complex was also inactivated by ATP in the presence of oligomycin B and carbonyl cyanide m-chlorophenylhydrazone. Ca2+ (which activates PDH phosphatase) and pyruvate or dichloroacetate (which inhibit PDH kinase) each increased the concentration of active PDH complex in a concentration-dependent manner in mitochondria oxidizing 2-oxoglutarate/L-malate. Values giving half-maximal activation were 10 nM-Ca2+, 3 mM-pyruvate and 16 microM-dichloroacetate. Activation by Ca2+ was inhibited by Na+ and Mg2+. Mitochondria incubated with [32P]Pi/2-oxoglutarate/L-malate incorporated 32P into three phosphorylation sites in the alpha-chain of PDH; relative rates of phosphorylation were sites 1 greater than 2 greater than 3, and of dephosphorylation, sites 2 greater than 1 greater than 3. Starvation ( 48h ) or induction of alloxan-diabetes had no effect on the total activity of PDH complex in skeletal-muscle mitochondria, but each decreased the concentration of active complex in mitochondria oxidizing 2-oxoglutarate/L-malate and increased the concentrations of Ca2+, pyruvate or dichloracetate required for half-maximal reactivation. In extracts of mitochondria the activity of PDH kinase was increased 2-3-fold by 48 h starvation or alloxan-diabetes, but the activity of PDH phosphatase was unchanged.     

Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate

1. The subcellular distribution of adenine nucleotides, acetyl-CoA, CoA, glutamate, 2-oxoglutarate, malate, oxaloacetate, pyruvate, phosphoenolpyruvate, 3-phosphoglycerate, glucose 6-phosphate, aspartate and citrate was studied in isolated hepatocytes in the absence and presence of glucagon by using a modified digitonin procedure for cell fractionation. 2. In the absence of glucagon, the cytosol contains about two-thirds of cellular ATP, some 40-50% of ADP, acetyl-CoA, citrate and phosphoenolpyruvate, more than 75% of total 2-oxoglutarate, glutamate, malate, oxaloacetate, pyruvate, 3-phosphoglycerate and aspartate, and all of glucose 6-phosphate. 3. In the presence of glucagon the cytosolic space shows an increase in the content of malate, phosphoenolpyruvate and 3-phosphoglycerate by more than 60%, and those of aspartate and glucose 6-phosphate rise by about 25%. Other metabolites remain unchanged. After glucagon treatment, cytosolic pyruvate is decreased by 37%, whereas glutamate and 2-oxoglutarate decrease by 70%. The [NAD(+)]/[NADH] ratios calculated from the cytosolic concentrations of the reactants of lactate dehydrogenase and malate dehydrogenase were the same. Glucagon shifts this ratio and also that of the [NADP(+)]/[NADPH] couple towards a more reduced state. 4. In the mitochondrial space glucagon causes an increase in the acetyl-CoA and ATP contents by 25%, and an increase in [phosphoenolpyruvate] by 50%. Other metabolites are not changed by glucagon. Oxaloacetate in the matrix is only slightly decreased after glucagon, yet glutamate and 2-oxoglutarate fall to about 25% of the respective control values. The [NAD(+)]/[NADH] ratios as calculated from the [3-hydroxybutyrate]/[acetoacetate] ratio and from the matrix [malate]/[oxaloacetate] couple are lowered by glucagon, yet in the latter case the values are about tenfold higher than in the former. 5. Glucagon and oleate stimulate gluconeogenesis from lactate to nearly the same extent. Oleate, however, does not produce the changes in cellular 2-oxoglutarate and glutamate as observed with glucagon. 6. The changes of the subcellular metabolite distribution after glucagon are compatible with the proposal that the stimulation of gluconeogenesis results from as yet unknown action(s) of the hormone at the mitochondrial level in concert with its established effects on proteolysis and lipolysis.     

Effects of ferulic acid on L-malate oxidation in isolated soybean mitochondria

The effects of ferulic acid on L-malate oxidation in mitochondria isolated from soybean (Glycine max L.) seedlings were investigated. Oxygen uptake and the products of L-malate oxidation were measured under two conditions: pH 6.8 and 7.8. At acidic pH, the activity of the NAD+-linked malic enzyme (L-malate:NAD+oxidoreductase [decarboxylating] EC 1.1.1.39) was favoured, whereas at alkaline pH a predominance of the L-malate dehydrogenase activity (L-malate:NAD+oxidoreductase EC 1.1.1.37) was apparent. Ferulic acid inhibited basal and coupled respiration during L-malate oxidation either at acidic or alkaline pH, reducing also the amounts of pyruvate or oxaloacetate produced. The results suggest that the site of ferulic acid action is situated at some step that precedes the respiratory chain. An interference with the L-malate entry into the mitochondria could be an explanation for the effects of ferulic acid, but the possibility of a direct inhibition of both enzymes involved in L-malate oxidation cannot be ruled out. This revised version was published online in August 2006 with corrections to the Cover Date.