Preparation and properties of rainbow trout liver mitochondria

Summary Mitochondria prepared from rainbow trout liver consistently display high respiratory control and ADP/O ratios. These appear to possess a complete Krebs cycle, since pyruvate, palmitoyll-carnitine, citrate, and various Krebs cycle intermediates can all be oxidized. Rapid oxidation of pyruvate and palmitoyll-carnitine requires the pressence of malate. Oxidation of palmitoyll-carnitine appears to inhibit pyruvate oxidation. Malate stimulates -ketoglutarate oxidation while aspartate inhibits glutamate oxidation, indicating the presence of malate--ketoglutarate and glutamate-aspartate carriers. These properties are compared with those of liver mitochondria from other species of fish.     

Acetyl-l-carnitine as a precursor of acetylcholine

Synthesis of [³H]acetylcholine from [³H]acetyl-l-carnitine was demonstrated in vitro by coupling the enzyme systems choline acetyltransferase and carnitine acetyltransferase. Likewise, both [³H] and [¹⁴C] labeled acetylcholine were produced when [³H]acetyl-l-carnitine andd-[U-¹⁴C] glucose were incubated with synaptosomal membrane preparations from rat brain. Transfer of the acetyl moiety from acetyl-l-carnitine to acetylcholine was dependent on concentration of acetyl-l-carnitine and required the presence of coenzyme A, which is normally produced as an inhibitory product of choline acetyltransferase. These results provide further evidence for a role of mitochondrial carnitine acetyltransferase in facilitating transfer of acetyl groups across mitochondrial membranes, thus regulating the availability in the cytoplasm of acetyl-CoA, a substrate of choline acetyltransferase. They are also consistent with a possible utility of acetyl-l-carnitine in the treatment of age-related cholinergic deficits.     

The effects of adenine nucleotides on pyruvate metabolism in rat liver

1. The effects of adenine nucleotides on pyruvate metabolism by isolated liver cells and isolated mitochondria have been investigated. The amount of pyruvate carboxylated has been estimated by determining the tricarboxylic acid-cycle intermediates, glutamate and aspartate accumulating in the incubation medium. The extent of pyruvate oxidation has been assessed by measuring oxygen uptake and the yield of ¹⁴CO₂ from [1-¹⁴C]pyruvate and [2-¹⁴C]pyruvate. 2. When catalytic amounts of adenine nucleotides (1–2mm) were added to suspensions of isolated liver cells incubated with pyruvate an ATP:ADP ratio greater than 6:1 was maintained. Both pyruvate oxidation to acetyl-CoA and the oxidation of acetyl-CoA through the tricarboxylic acid cycle were stimulated but pyruvate carboxylation was not affected. The production of acetyl-CoA exceeded the capacity of the cells for the oxidation of acetyl-CoA and the excess was converted into ketone bodies. 3. If a low ATP:ADP ratio was maintained in isolated cells or mitochondria by incubating them with dinitrophenol or hexokinase, pyruvate carboxylation was grossly inhibited, oxygen uptake depressed and ketone-body formation stimulated. Measurement of oxaloacetate concentrations confirmed that under these conditions oxaloacetate was rate-limiting for the oxidation of acetyl-CoA via the tricarboxylic acid cycle. The inclusion in the incubation medium of fumarate (1·25mm) completely prevented the ketogenic action of dinitrophenol or hexokinase. 4. When ADP (5mm) was added to a suspension of isolated liver cells incubated with pyruvate an actual ADP concentration of about 1mm was attained. This brought about effects on pyruvate metabolism similar to those obtained with dinitrophenol or hexokinase. 5. These results support the concept that the relative concentrations of adenine nucleotides within the liver cell may play a role in governing the rates of pyruvate oxidation and carboxylation. In addition, they provide further evidence that the availability of oxaloacetate in the liver cell can play a key role in determining whether acetyl-CoA arising from pyruvate is oxidized through the tricarboxylic acid cycle or converted into ketone bodies.     

Acetyl-coenzyme a can regulate activity of the mitochondrial pyruvate dehydrogenase complex in situ

In vitro, the pyruvate dehydrogenase complex is sensitive to product inhibition by NADH and acetyl-coenzyme A (CoA). Based upon K_m and K_i relationships, it was suggested that NADH can play a primary role in control of pyruvate dehydrogenase complex activity in vivo (JA Miernyk, DD Randall [1987] Plant Physiol 83:306-310). We have now extended the in vitro studies of product inhibition by assaying pyruvate dehydrogenase complex activity in situ, using purified intact mitochondria from green pea (Pisum sativum) seedlings. In situ activity of the pyruvate dehydrogenase complex is inhibited when mitochondria are incubated with malonate. In some instances, isolated mitochondria show an apparent lack of coupling during pyruvate oxidation. The inhibition by malonate, and the apparent lack of coupling, can both be explained by an accumulation of acetyl-CoA. Inhibition could be alleviated by addition of oxalacetate, high levels of malate, or l-carnitine. The CoA pool in nonrespiring mitochondria was approximately 150 micromolar, but doubled during pyruvate oxidation, when 60 to 95% of the total was in the form of acetyl-CoA. Our results indicate that in situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition.     

The effect of cosubstrates on tricarboxylic acid cycle dynamics during pyruvate oxidation: the formation of alpha-ketoglutarate and utilization of glutamate by mitochondria from rabbit brain.

—Data comparing tricarboxylic acid cycle dynamics in mitochondria from rabbit brain using [2- or 3-¹⁴C]pyruvate with and without cosubstrates (malate, α-ketoglutarate, glutamate) are reported. With a physiological concentration of an unlabelled cosubstrate, from 90-99% of the isotope remained in cycle intermediates. However, the liberation of ¹⁴CO₂ and the presence of ¹⁴C in the C-1 position of α-ketoglutarate indicated that multiple turns of the cycle occurred. Entry of pyruvate into the cycle was greater with malate than with either α-ketoglutarate or glutamate as cosubstrate. With malate as cosubstrate for [¹⁴C]pyruvate the amount of [¹⁴C]citrate which accumulated averaged 30nmol/ml or 23% of the pyruvate utilized while α-ketoglutarate averaged 45 nmol/ml or 35% of the pyruvate utilized. With α-ketoglutarate as cosubstrate for [¹⁴C]pyruvate, the average amount of [¹⁴C]citrate which accumulated decreased to 8 nmol/ml or 10% of the pyruvate utilized while [¹⁴C]α-ketoglutarate increased slightly to 52 nmol/ml or an increase to 62%, largely due to a decrease in pyruvate utilization. The percentage of ¹⁴C found in α-ketoglutarate was always greater than that found in malate, irrespective of whether α-ketoglutarate or malate was the cosubstrate for either [2- or 3-¹⁴C]pyruvate. The fraction of ¹⁴CO₂ produced was slightly greater with α-ketoglutarate as cosubstrate than with malate. This observation and the fact that malate had a higher specific activity than did α-ketoglutarate when α-ketoglutarate was the cosubstrate, indicated a preferential utilization of α-ketoglutarate formed within the mitochondria. When l -glutamate was a cosubstrate for [¹⁴C]pyruvate the principal radioactive product was glutamate, formed by isotopic exchange of glutamate with [¹⁴C] α-ketoglutarate. If malate was also added, [¹⁴C]citrate accumulated although pyruvate entry did not increase. Due to retention of isotope in glutamate, little [¹⁴C]succinate, malate or aspartate accumulated. When [U-¹⁴C]l -glutamate was used in conjunction with unlabelled pyruvate more ¹⁴C entered the cycle than when unlabelled glutamate was used with [¹⁴C]pyruvate and led to α-ketoglutarate, succinate and aspartate as the major isotopic products. When in addition, unlabelled malate was added, total and isotopic α-ketoglutarate increased while [¹⁴C]aspartate decreased. The increase in [¹⁴C]succinate when [¹⁴C] glutamate was used indicated an increase in the flux through α-ketoglutarate dehydrogenase and was accompanied by a decrease of pyruvate utilization as compared to experiments when either α-ketoglutarate or glutamate were present at low concentration. It is concluded that the tricarboxylic acid cycle in brain mitochondria operates in at least three open segments, (1) pyruvate plus malate (oxaloacetate) to citrate; (2) citrate to α-ketoglutarate and; (3) α-ketoglutarate to malate, and that at any given time, the relative rates of these segments depend upon the substrate composition of the environment of the mitochondria. These data suggest an approach to a steady state consistent with the kinetic properties of the tricarboxylic acid cycle within the mitochondria.     

Cyclic AMP induced inhibition of pyruvate kinase flux in the intact liver cell.

The rate of pyruvate kinase flux in the intact cell is estimated by a new procedure, involving trapping of ¹⁴C from NaH¹⁴CO₃ in a large pyruvate + lactate pool, and calculation of the specific activity of phosphoenol pyruvate. With high concentrations of pyruvate as substrate for isolated rat liver cells, cyclic AMP (0.1 mM) depresses pyruvate kinase flux by about 45%, in addition to inhibiting both glucose and lactate formation. The inhibition of pyruvate kinase may cause an inhibition of hydrogen translocation from the mitochondria to the cytosol.     

The effect of 2-oxoglutarate or 3-hydroxybutyrate on pyruvate dehydrogenase complex in isolated cerebro-cortical mitochondria

The oxidation of pyruvate is mediated by the pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12 and EC 1.6.4.3) whose catalytic activity is influenced by phosphorylation and by product inhibition. 2-Oxoglutarate and 3-hydroxybutyrate are readily utilized by brain mitochondria and inhibit pyruvate oxidation. To further elucidate the regulatory behavior of brain PDHC, the effects of 2-oxoglutarate and 3-hydroxyburyrate on the flux of PDHC (as determined by [1-¹⁴C]pyruvate decarboxylation) and the activation (phosphorylation) state of PDHC were determined in isolated, non-synaptic cerebro-cortical mitochondria in the presence or absence of added adenine nucleotides (ADP or ATP). [1-¹⁴C]Pyruvate decarboxylation by these mitochondria is consistently depressed by either 3-hydroxybutyrate or 2-oxoglutarate in the presence of ADP when mitochondrial respiration is stimulated. In the presence of exogenous ADP, 3-hydroxybutyrate inhibits pyruvate oxidation mainly through the phosphorylation of PDHC, since the reduction of the PDHC flux parallels the depression of PDHC activation state under these conditions. On the other hand, in addition to the phosphorylation of PDHC, 2-oxoglutarate may also regulate pyruvate oxidation by product inhibition of PDHC in the presence of 0.5 mM pyruvate plus ADP or 5 mM pyruvate alone. This conclusion is based upon the observation that 2-oxoglutarate inhibits [1-¹⁴C]pyruvate decarboxylation to a much greater extent than that predicted from the PDHC activation state (i.e. catalytic capacity) alone. In conjunction with the results from our previous study (Lai, J. C. K. and Sheu, K.-F. R. (1985) J. Neurochem. 45, 1861–1868), the data of the present study are consistent with the notion that the relative importance of the various mechanisms that regulate brain and peripheral tissue PDHCs shows interesting differences.     

Regulation of glutamine and pyruvate oxidation in cultured adrenocortical cells by cortisol,antioxidants, and oxygen: Effects on cell proliferation

The regulation of CO₂ production from [U-¹⁴C]glutamine and C₂ of [2-¹⁴C]pyruvate was investigated in cultured bovine adrenocortical cells, and the effect of alterations in the relative rates of oxidation of these substrates on cell proliferation, particularly in the presence of an inhibitor of transamination reactions, was examined. ¹⁴CO₂ production from 2 mM [U-¹⁴C]glutamine and 2 mM [2-¹⁴C]pyruvate was measured in the presence of 100 μM 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. Treatment of primary cultures for 24 h with 50 μM cortisol increased the oxidation of [¹⁴C]glutamine relative to that of [¹⁴C]pyruvate, an effect dependent on prior low cell density. Cortisol treatment also resulted in a prolonged delay in the onset of proliferation from low density, and completely inhibited growth in the presence of 2 mM aminooxyacetate, which reduces mitochondrial utilization of glutamine. The effects on glutamine and pyruvate metabolism and on cell growth, with or without aminooxyacetate, were prevented by simultaneous treatment with the antioxidants dimethyl sulfoxide (10 mM) and butylated hydroxyanisole (100 μM), suggesting the involvement of lipid peroxidation in the action of cortisol, as previously demonstrated for its action on 11β-hydroxylase. During continued proliferation of adrenocortical cells in the absence of cortisol there was also a slower increase in the oxidation of [¹⁴C]glutamine relative to that of [¹⁴C]pyruvate as a function of population doubling level. The rate of this increase was slowed by growth of cells in 2% O₂ rather than the standard 19% O₂, and accelerated by continued growth of cells in the presence of cortisol. The rate of increase in the oxidation of [¹⁴C]glutamine relative to that of [¹⁴C]pyruvate under these three conditions correlated with inhibition of cell growth by aminooxyacetate. In contrast to the complete inhibition of growth in aminooxyacetate demonstrated by cortisol-treated cells, control cells (19% O₂) did proliferate, although growth was limited, whereas cells at 2% O₂ proliferated to a much greater extent. In the absence of aminooxyacetate the rate of growth in primary adrenocortical cell cultures under these three conditions was similar. Lipid peroxidation appears to make cultured adrenocortical cells dependent on glutamine for mitochondrial function and proliferation by inhibiting the utilization of the normal substrate, pyruvate.     

Studies on the regulation of leucine catabolism: Mechanism responsible for oxidizable substrate inhibition and dichloroacetate stimulation of leucine oxidation by the heart

In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1-¹⁴C]leucine to ¹⁴CO₂ at a rapid rate and releases only small amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1-¹⁴C]leucine oxidation, α-[1-¹⁴C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1-¹⁴C]leucine oxidation to ¹⁴CO₂ and the release of large amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1-¹⁴C]leucine oxidation, decreases α-[1-¹⁴C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1-¹⁴C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex.     

Studies on brain-cortex slices: Differences in the oxidation of 14C-labelled glucose and pyruvate revealed by the action of triethyltin and other toxic agents

1. The rate of appearance of ¹⁴CO₂ from [6-¹⁴C]glucose and [3-¹⁴C]pyruvate was measured. Pyruvate is oxidized to carbon dioxide twice as fast as glucose, although the oxygen uptake is almost the same with each substrate. 2. The presence of 30μm-2,4-dinitrophenol increases the output of ¹⁴CO₂ from [6-¹⁴C]glucose sixfold whereas the oxygen uptake is not quite doubled. Similar results are obtained with 0·1m-potassium chloride. The stimulating action of these two agents on the output of ¹⁴CO₂ from [3-¹⁴C]pyruvate is much less than on that from [6-¹⁴C]glucose. 3. The effects of oligomycin, ouabain and triethyltin on the respiration of control and stimulated brain-cortex slices were studied. Triethyltin (1·3μm) inhibited the oxidation of [6-¹⁴C]glucose more than 70%, but did not inhibit the oxidation of[3-¹⁴C]pyruvate. [3-¹⁴C]pyruvate. 4. The production of lactic acid by brain-cortex slices incubated with glucose is twice as great as that with pyruvate. Lactic acid increases two and a half times in the presence of either triethyltin or oligomycin when the substrate is glucose, but is no different from the control when the substrate is pyruvate. 5. With kidney slices the production of lactic acid from glucose is very low. It is increased by oligomycin but not by triethyltin. 6. The results are discussed in terms of the oxidation of the extramitochondrial NADH₂ produced during glycolysis.     

Effect of tolbutamide on the metabolism of Tetrahymena

Tolbutamide partially inhibited the growth but increased the glycogen content of Tetrahymena pyriformis in logarithmically growing cultures. Tolbutamide slightly increased ¹⁴CO² production from [1-¹⁴C] and [6-¹⁴HC] glucose and [2-¹⁴C] pyruvate, but had little effect on the oxidation of [1-¹⁴C] acetate when any of these substrates were added to the proteose-peptone medium in which the cells had been grown. Measurement of ¹⁴CO₂ production from [1-¹⁴C] and [2-^I4C]-glyoxylate showed that this substrate was primarily oxidized via the glyoxylate cycle, with little if any oxidation occurring via the peroxisomal glyoxylate oxidase. Addition of tolbutamide inhibited the glyoxylate cycle as indicated by a marked reduction in label appearing in CO₂ and in glycogen from labeled acetate. In control cells, addition of acetate strongly inhibited the oxidation of [2-¹⁴C]-pyruvate whereas addition of pyruvate had little effect on the oxidation of [1-¹⁴C]-acetate. Acetate was more effective than pyruvate in preventing the growth inhibitory and glycogen-increasing effects of tolbutamide. The data suggest that one effect of tolbutamide may be to interfere with the transfer of isocitrate and acetyl CoA across mitochondrial membranes.     

Characterization of oxalate as a catabolite of dichloroacetate responsible for the inhibition of gluconeogenesis and pyruvate carboxylation in rat liver cells

In isolated hepatocytes, dichloroacetate decreased glucose synthesis from lactate, pyruvate and alanine, but not from substrates which bypass pyruvate carboxylase (propionate, glycerol). It was also found to inhibit pyruvate carboxylation in isolated mitochondria, but only after a preincubation period, and had no effect on partially purified pyruvate carboxylase. Hepatocytes and liver mitochondria metabolized [¹⁴C] dichloroacetate to oxalate which inhibits pyruvate carboxylase and mimics, without preincubation, the effects of dichloroacetate in mitochondrial pyruvate carboxylation. Thus, oxalate appears to be responsible for the inhibition of gluconeogenesis by dichloroacetate at the level of pyruvate carboxylation.     

Stimulation of flux through hepatic pyruvate dehydrogenase by 3-mercaptopicolinate

Isolated hepatocytes from 24-h-starved rats were used to assess the possible effect of Ahe hypoglycaemic agent 3-mercaptopicolinate on flux through the hepatic pyruvate dehydrogenase complex. Increasing the extraceIIular pyruvate concentration from 1 mM to 2 mM or 5 mM resulted in an increase in flux through pyruvate dehydrogenase and the tricarboxylic acid cycle as measured by¹⁴CO₂ evolution from [1-¹⁴C]pyruvate and [3-¹⁴C]pyruvate. Gluconeogenesis was inhibited by 3-mercaptopicolinate from both 1 mM and 2 mM pyruvate, but significant increases in malate and citrate concentrations only occurred in cells incubated with 1 mM pyruvate. Flux through pyruvate dehydrogenase was stimulated by 3-mercaptopicolinate with 1 mM pyruvate but was unaltered with 2 mM pyruvate. Dichloroacetate stimulated flux through pyruvate dehydrogenase with no effect on gluconeogenesis in the presence of I mM pyruvate. There was no effect of 3-mercaptopicolinate, administered in vivo, to 24-h-starved rats on the activity of pyruvate dehydrogenase in freeze-clamped heart or liver tissue, although the drug did decrease blood glucose concentration and increase the blood concentrations of lactate and alanine. Dichloroacetate, administered in vivo to 24-h-starved rats, increased the activity of pyruvate dehydrogenase in freeze-clamped heart and liver, and caused decreases in the blood concentrations of glucose, lactate , and alanine. The results suggest that 3-mercaptopicolinate increases flux through hepatocyte pyruvate dehydrogenase by an indirect mechanism.     

The role of the Krebs cycle in the generation of intramitochondrial reducing equivalents for the 11 beta-hydroxylation of deoxycorticosterone in isolated rat adrenal cells

Isolated rat adrenal cells were used to study the possible pathways of intramitochondrial NADPH generation for 11β-hydroxylation of 11-deoxycorticosterone. Pyruvate was efficiently utilized by the mitochondria as shown by evolution of ¹⁴CO₂ from [1-¹⁴C]- and [2-¹⁴C]pyruvate. Citrate, isocitrate, succinate, and malate were not utilized by intact cells due to their inability to permeate the plasma membrane. For every mole of corticosterone formed, 1.9 and 0.8 moles of ¹⁴CO₂ were formed from [1-¹⁴C]- and [2-¹⁴C]pyruvate, respectively, indicating that pyruvate dehydrogenase was quite active and supplied acetyl C?oA to the Krebs cycle. Fluorocitrate and 2,4-dinitrophenol inhibited 11β-hydroxylation of 11-deoxycorticosterone as well as the production of ¹⁴CO₂ from [2-¹⁴C]pyruvate. Comparison of data with the two inhibitors showed that for the same percentage of inhibition of ¹⁴CO₂ production, the inhibition of 11β-hydroxylation was greater with 2,4-dinitrophenol than with fluorocitrate. It is concluded that operation of the Krebs cycle may be essential for 11β-hydroxylation to occur primarily because NADH generated by the cycle provides ATP, via the respiratory chain, as well as the substrate for the energy-linked transhydrogenase that forms NADPH. The NADPH required for 11β-hydroxylation seems to be derived to a large extent via the energy-linked transhydrogenase.     

N-methyl oxidation in liver mitochondria of triiodothyronine-treated and thyroidectomized rats

The levels of sarcosine dehydrogenase and acid-nonextractable flavin in the inner matrix of mitochondria of rat liver are decreased in animals treated with triiodothyronine and are elevated in the mitochondria obtained from thyroidectomized animals. Administration of triiodothyronine does not affect the electron-transfer flavoprotein associated with the sarcosine dehydrogenase or the relative amounts of soluble and membrane-bound proteins of the mitochondria. In phosphate-washed mitochondria from either the controls or the triiodothyronine-treated rats, the O₂ uptake equals the total of the [¹⁴C]formaldehyde and [β-¹⁴C]serine isolated as reaction products of the sarcosine-[¹⁴C]methyl group. In contrast to its restraint of sarcosine or choline oxidation in preparations capable of oxidative phosphorylation, ADP does not inhibit the oxidation of these substrates in mitochondria of rats given triiodothyronine.     

A 14CO2 ratios method for detecting pyruvate carboxylation

The pattern of oxidative metabolism of pyruvate may be assessed by comparing the steady-state 14CO2 production from four isotopes in identical samples. The assay requires measuring the ratios of steady-state 14CO2 production from two isotope pairs, [2-14C]pyruvate:[3-14C]pyruvate and [1-14C]acetate:[2-14C]acetate. These ratios are defined as the "pyruvate 14CO2 ratio" and the "acetate 14CO2 ratio," respectively. If pyruvate is metabolized exclusively via pyruvate dehydrogenase (PDH), the two ratios will be identical. Alternatively, if any pyruvate enters the tricarboxylic acid (TCA) cycle via pyruvate carboxylation (PC), the pyruvate 14CO2 ratio will be less than the acetate 14CO2 ratio. If pyruvate enters the TCA cycle only through PC (with oxaloacetate and fumarate in equilibrium) the pyruvate 14CO2 ratio will approach a value of 1.0. An equation is presented for the quantitative evaluation of pyruvate oxidation by these two pathways. We have used this method to detect relative changes in the pattern of pyruvate metabolism in rat liver mitochondria produced by exposure to 1 mM octanoyl carnitine, a compound known to alter the PC:PDH activity ratio. The major advantages of the method are (i) that it provides a sensitive method for detecting pyruvate carboxylation at physiological pyruvate concentrations and (ii) that it provides a method for distinguishing between effects on pyruvate transport and effects on pyruvate oxidation.     

Catabolism of 2-methyloctanoic acid and 3beta-hydroxycholest-5-en-26-oic acid

1. 2-Methyl[1-¹⁴C]octanoic acid was synthesized from 2-bromo-octane and ¹⁴CO₂. 2. 2-Methyl[1-¹⁴C]octanoic acid was readily oxidized to propionic acid and carbon dioxide by mitochondrial preparations from liver, less readily oxidized by adrenal and kidney (mitochondria), and only poorly oxidized by heart, spleen and brown fat (mitochondria). 3. 3β-Hydroxy[26-¹⁴C]cholest-5-en-26-oic acid was rapidly oxidized by mammalian-liver mitochondria to propionic acid and carbon dioxide. Caiman-liver and toad-liver mitochondria also oxidized this steroid acid. 4. The oxidation of propionic acid, octanoic acid and palmitic acid by mitochondrial preparations from these various tissues was also studied. 5. Added carnitine did not stimulate 2-methyloctanoic acid oxidation and feebly stimulated 3β-hydroxycholest-5-en-26-oic acid oxidation. 6. The significance of these results is discussed in relation to sterol catabolism in mammals and non-mammalian species.     

Inhibition of anion transport across the mitochondrial membrane by amytal

It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the K_i value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p-trifluoromethoxyphenylhydrazone.Using [¹⁴C] succinate and [¹⁴C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/P_i, succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited P_i transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [¹⁴C]succinate/anion (P_i, succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and P_i translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and P_i translocation across the mitochondrial membrane.     

Oxalate synthesis from [14C1]glycollate and [14C1]glycoxylate in the hepatectomized rat

Hepatectomy significantly altered the metabolism of [1-¹⁴C]glyoxylate and [1-¹⁴C]glycollate in the rat. The production of ¹⁴CO₂ was reduced by 47% and 77%–86%, respectively, indicating the involvement of the liver in the oxidation of both substrates. Unidentified intermediates, assumed to be primary glycine, serine and ethanolamine, were also reduced by over 50%, was would be expected from the removal of the aminotransferase enzymes through the hepatectomy. The biosynthesis of [¹⁴C]oxalate from [1-¹⁴C]glycollate was reduced by more than 80% in the hepatectomized rat. This suggests that this oxidation is primarily catalyzed by the liver enzymes, glycolic acid oxidase and glycolic acid dehydrogenase, in the intact rat. The limited formation of [¹⁴C]oxalate from [¹⁴₁]glycollate observed in the hepatectomized rat is probably catalyzed by lactate dehydrogenase or extrahepatic glycolic acid oxidase. Hepatectomy did not significantly alter the rate of formation of [¹⁴C]oxalate from [¹⁴₁]glyoxylate. However, since saturating concentrations of glyoxylate could not be used because of the toxicity of this substrate, the involvement of glycollic acid oxidase in this oxidation reaction in the intact rat can not be ruled out. In the hepatectomized rat, lactate dehydrogenase appears to be the enzyme making the major contribution, although other as yet not identified enzymes may be contributing. The increased deposition of oxalate in the tissues, oxalosis, may result from the shift in oxalate synthesis from the liver to the extrahepatic tissues.     

Stimulation of pyruvate dehydrogenase activity by insulin-dextran complex in mouse adipose tissue.

Soluble and stable insulin-dextran complex was prepared. Pyruvate dehydrogenase activity, as assayed by ¹⁴CO₂ formation from [1-¹⁴C]-pyruvate in crude mitochondria of mouse adipose tissue, was increased after incubation of fat pads with native insulin or insulin-dextran. The direct addition of insulin or insulin-dextran to mitochondria was without effect. At submaximal stimulation, insulin-dextran was 10 times less effective than native insulin but the degree of maximal stimulation and the time course of activation by insulin and insulin-dextran were similar. The results favor the concept that the activation of pyruvate dehydrogenase in fat cells does not need the entry of insulin into cells.