The nature and control of the tricarboxylate cycle in beetle flight muscle.

The only exogenous substrates oxidized by mitochondria isolated from the flight muscle of the Japanese beetle (Popillia japonica) are proline, pyruvate and glycerol 3-phosphate. The highest rate of oxygen consumption is obtained with proline. The oxidation of proline leads to the production of more NH3 than alanine, indicating a functioning glutamate dehydrogenase (EC 1.4.1.2). Studies of mitochondrial extracts confirm the presence of a very active glutamate dehydrogenase, and this enzyme is found to be activated by ADP and inhibited by ATP. These extracts also show high alanine aminotransferase activity (EC 2.6.1.2) and a uniquely active "malic'' enzyme (EC 1.1.1.39). The "malic'' enzyme is activated by succinate and inhibited by ATP and by pyruvate. It is suggested that the input of tricarboxylate-cycle intermediate from proline oxidation is balanced by the formation of pyruvate from malate, and the complete oxidation of the majority of the pyruvate. Studies of the steady-state concentrations of mitochondrial CoASH and CoA thioesters during proline oxidation show a high succinyl (3-carboxypropionyl)-CoA content which falls on activating respiration with ADP. There is a concomitant rise in CoASH. However, the reverse transition, from state-3 to state-4 respiration, causes only very slight changes in acylation. The reasons for this are discussed. Studies of the mitochondrial content of glutamate, 2-oxoglutarate, malate, pyruvate, citrate and isocitrate during the same phases of proline oxidation give results consistent with control at the level of glutamate dehydrogenase and isocitrate dehydrogenase during proline oxidation, with the possibility of further control at "malic'' enzyme. During the oxidation of pyruvate all of the tricarboxylate-cycle intermediates and NAD(P)H follow the pattern of changes described in the blowfly (Johnson & Hansford, 1975; Hansford, 1974) and isocitrate dehydrogenase is identified as the primary site of control.?2OAuthor     

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.     

The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The oxidized and reduced nicotinamide-adenine dinucleotide content of flight muscle and isolated mitochondria, the adenosine triphosphate and adenosine diphosphate content of mitochondria, and the energy status of the mitochondria during controlled respiration.

1. A study is presented of the mitochondrial NADH content during controlled (state 4) and active (state 3) pyruvate oxidation by blowfly flight-muscle mitochondria. The results confirm and extend those of an earlier study (Hansford, 1972), which indicated an increased reduction in state 3. Nicotinamide nucleotide is normally highly oxidized during state 4; however, there can be substantial reduction in the presence of carnitine or high concentrations of proline, or on lengthy incubation in the presence of either of the systems used to generate intramitochondrial tricarboxylate-cycle intermediate. 2. Omission of phosphate leads to substantial reduction and this can be reversed by adding phosphate or acetate. 3. Estimations of NAD-+ and NADH in fly thoraces show a marked increase in NADH on flight, tending to corroborate the results of mitochondrial experiments and testifying to the importance of dehydrogenase activation in this tissue. 4. Determination of intramitochondrial adenine nucleotides reveals a total of 4-5 nmol/mg of protein, and an ADP content of less than 0.1 nmol/mg during state 4 oxidation of pyruvate and proline. ATP content is found to increase slowly during state 4 and this is attributed to the net phosphorylation of AMP. 5. The uncoupling agent carbonyl cyanide p=trifluoromethoxyphenylhydrazone leads to hydrolysis of some, but not all, of the mitochondrial ATP. Studies of mitochondrial ATPase (adenosine triphosphatase), measured by external pH change, show that it is inactive unless the mitochondria are allowed to respire for several minutes in state 4 in the presence of phosphate before the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It is suggested that phosphate uptake is essential for maximal ATPase activity. 6. Studies of the fluorescence of the fluorochrome 8-anilino-1-naphthalensulphonic acid suggest that the energy status of the mitochondrion is high during state 4-pyruvate oxidattion, and decrease slightly in state 3. The implications of these findings are discussed.     

Regulation of pyruvate oxidation in blowfly flight muscle mitochondria: Requirement for ADP

Blowfly (Phormia regina) flight muscle mitochondria oxidized pyruvate (+ proline) in the presence of either ADP (coupled respiration) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP-uncoupled respiration). There was an absolute requirement for ADP (K_m = 8.0 μm) when pyruvate oxidation was stimulated by FCCP in the presence of oligomycin. This requirement for ADP was limited to the oxidation of pyruvate; uncoupled α-glycerolphosphate oxidation proceeded maximally even in the absence of added ADP. Atractylate inhibited uncoupled pyruvate oxidation whether added before (>99%) or after (95%) initiation of respiration with FCCP. In the presence of FCCP, oligomycin, and limiting concentrations of ADP (less than 110 μm), there was a shutoff in the uptake of oxygen. This inhibition of respiration was completely reversed by the addition of more ADP. Plots of net oxygen uptake as a function of the limiting ADP concentration were linear; the observed ADP/O ratio was 0.22 ± 0.025. An ADP/O ratio of 0.2 was predicted if phosphorylation occurred only at the succinyl-CoA synthetase step of the tricarboxylate cycle. Experiments performed in the presence of limiting concentrations of ADP, and designed to monitor changes in the mitochondrial content of ADP and ATP, demonstrated that the shutoff in oxygen uptake was not due to the presence of a high intramitochondrial concentration of ATP. Indeed, ATP, added to the medium prior to the addition of FCCP, inhibited uncoupled pyruvate oxidation; the apparent K_I was 0.8 mm. These results are consistent with the hypothesis that it is the intramitochondrial ATP/ADP ratio that is one of the controlling factors in determining the rate of flux through the tricarboxylate cycle. Changes in the mitochondrial content of citrate, isocitrate, α-ketoglutarate, and malate during uncoupled pyruvate oxidation in the presence of a limiting concentration of ADP were consistent with the hypothesis that the mitochondrial NAD⁺-linked isocitric dehydrogenase is a major site for such control through the tricarboxylate cycle.     

Some properties of mitochondria isolated from the flight muscle of the periodical cicada, Magicicada septendecim

Mitochondria from the flight muscle of the periodical cicada oxidize pyruvate and d-glycerol 1-phosphate at rates comparable with those obtained with flight-muscle mitochondria from other insects. The oxidation of d-glycerol 1-phosphate is greatly stimulated by low concentrations of Ca(2+). However, oxidative phosphorylation with this substrate is optimum over only a narrow range of Ca(2+) concentration, because of the ability of these mitochondria actively to accumulate Ca(2+) present at micromolar concentrations. The oxidation of pyruvate via the complete tricarboxylic acid cycle is enhanced by high concentrations of phosphate. When both pyruvate and d-glycerol 1-phosphate are present simultaneously, there is no simple summation of the rates obtained with the substrates singly. Acetyl-l-carnitine, palmitoyl-l-carnitine, glutamate and 2-oxoglutarate are oxidized at rates similar to those obtained with mammalian mitochondria, though lower than those obtained with the two prime substrates. However, no other tricarboxylic acid-cycle intermediates added to the medium were oxidized. From these and other observations it has been concluded that these mitochondria possess a previously undescribed combination of substrate-anion permeases.     

Isolation and properties of flight muscle mitochondria of the bumblebee Bombus terrestris (L.)

This report describes the isolation procedure and properties of tightly coupled flight muscle mitochondria of the bumblebee Bombus terrestris (L.). The highest respiratory control index was observed upon oxidation of pyruvate, whereas the highest respiration rates were registered upon oxidation of a combination of the following substrates: pyruvate + malate, pyruvate + proline, or pyruvate + glutamate. The respiration rates upon oxidation of malate, glutamate, glutamate + malate, or succinate were very low. At variance with flight muscle mitochondria of a number of other insects reported earlier, B. terrestris mitochondria did not show high rates of respiration supported by oxidation of proline. The maximal respiration rates were observed upon oxidation of α-glycerophosphate. Bumblebee mitochondria are capable of maintaining high membrane potential in the absence of added respiratory substrates, which was completely dissipated by the addition of rotenone, suggesting high amount of intramitochondrial NAD-linked oxidative substrates. Pyruvate and α-glycerophosphate appear to be the optimal oxidative substrates for maintaining the high rates of oxidative metabolism of the bumblebee mitochondria.     

Changes in the contents of adenine nucleotides and intermediates of glycolysis and citric acid cycle in flight muscle of the locust upon flight and their relationship to the control of the cycle

1. The contents of some intermediates of glycolysis, the citric acid cycle and adenine nucleotides have been measured in the freeze-clamped locust flight muscle at rest and after 10s and 3min flight. The contents of glucose 6-phosphate, pyruvate, alanine and especially fructose bisphosphate and triose phosphates increased markedly upon flight. The content of acetyl-CoA is decreased after 3min flight whereas that of acetylcarnitine is decreased markedly after 10s flight, but returns towards the resting value after 3min flight. The content of citrate is markedly decreased after both 10s and 3min flight, whereas that of isocitrate is changed very little after 10s and is increased by 50% after 3min. The content of oxaloacetate is very low in insect flight muscle and hence it was measured by a sensitive radiochemical assay. The content of oxaloacetate increased about 2-fold after 3min flight. A similar change was observed in the content of malate. The content of ATP decreased about 15%, whereas those of ADP and AMP increased about 2-fold after 3min flight. 2. Calculations based on O(2) uptake of the intact insect indicate that the rate of the citric acid cycle must be increased >100-fold during flight. Consequently, if citrate synthase catalyses a non-equilibrium reaction, the activity of the enzyme must increase >100-fold during flight. However, changes in the concentrations of possible regulators of citrate synthase, oxaloacetate, acetyl-CoA and citrate (which is an allosteric inhibitor), are not sufficient to account for this change in activity. It is concluded that there may be much larger changes in the free concentration of oxaloacetate than are indicated by the changes in the total content of this metabolite or that other unknown factors must play an additional role in the regulation of citrate synthase activity. 3. The increased content of oxaloacetate could be produced via pyruvate carboxylase, which may be stimulated during the early stages of flight by the increased concentration of pyruvate. 4. The decreases in the concentrations of citrate and alpha-oxoglutarate indicate that isocitrate dehydrogenase and oxoglutarate dehydrogenase may be stimulated by factors other than their pathway substrates during the early stages of flight. 5. Calculated mitochondrial and cytosolic NAD(+)/NADH ratios are both increased upon flight. The change in the mitochondrial ratio indicates the importance of the intramitochondrial ATP/ADP concentration ratio in the regulation of the rate of electron transfer in this muscle.     

Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria

1. High rates of state 3 pyruvate oxidation are dependent on high concentrations of inorganic phosphate and a predominance of ADP in the intramitochondrial pool of adenine nucleotides. The latter requirement is most marked at alkaline pH values, where ATP is profoundly inhibitory. 2. Addition of CaCl(2) during state 4, state 3 (Chance & Williams, 1955) or uncoupled pyruvate oxidation causes a marked inhibition in the rate of oxygen uptake when low concentrations of mitochondria are employed, but may lead to an enhancement of state 4 oxygen uptake when very high concentrations of mitochondria are used. 3. These properties are consistent with the kinetics of the NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from this tissue, which is activated by isocitrate, citrate, ADP, phosphate and H(+) ions, and inhibited by ATP, NADH and Ca(2+). 4. Studies of the redox state of NAD and cytochrome c show that addition of ADP during pyruvate oxidation causes a slight reduction, whereas addition during glycerol phosphate oxidation causes a ;classical' oxidation. Nevertheless, it is concluded that pyruvate oxidation is probably limited by the respiratory chain in state 4 and by the NAD-linked isocitrate dehydrogenase in state 3. 5. The oxidation of 2-oxoglutarate by swollen mitochondria is also stimulated by high concentrations of ADP and phosphate, and is not uncoupled by arsenate.     

Pyruvate metabolism in rat brain mitochondria.

1. Oxidation of pyruvate by rat brain mitochondria was stimulated in state 3 by malate or succinate up to 250 nmoles O2/mg protein/min. Oxidation of malate, succinate, 2-oxoglutarate or glutamate as the sole substrates, was 1/4 - 1/5 that observed with pyruvate. 2. Maximum oxygen consumption in state 3 was observed at pH 6.90 - 7.20, whereas in state 4 it was not affected by changes in pH. 3. In state 4, in the absence of exogenous acceptor or acetyl residues, acetate was the main oxidation product, corresponding to about 80% of the amount of pyruvate utilized. Malate did not affect the rate of pyruvate utilization but lowered acetate concentration and raised concentration of citrate and 2-oxoglutarate. 4. In state 3, pyruvate and malate were converted mainly to 2-oxoglutarate, its concentration being three times as high as that of citrate. 5. Formation of citrate, 2-oxoglutarate and acetate from pyruvate in brain is considered as a function of availability of the acceptor of acetyl residues and the energy state of mitochondrion.     

Specificity and locale of the l-3-glycerophosphate–flavoprotein oxidoreductase of mitochondria isolated from the flight muscle of Sarcophaga barbata Thoms

1. The oxidation of l-3-glycerophosphate by flight-muscle mitochondria isolated from the flesh fly Sarcophaga barbata has been studied. Use of substrate analogues indicates that the catalytic and effector l-3-glycerophosphate binding sites on the allosteric l-3-glycerophosphate-flavoprotein oxidoreductase differ markedly in specificity. 2. The l-3-glycerophosphate-cyanoferrate oxidoreductase system in these mitochondria is antimycin-insensitive whereas the corresponding NADH-cyanoferrate oxidoreductase is extremely sensitive to this respiratory-chain inhibitor. Also no swelling is observed when these mitochondria are suspended in iso-osmotic solutions of ammonium glycerophosphate in contrast with the extensive swelling seen in similar solutions of ammonium pyruvate. These observations indicate that l-3-glycerophosphate does not penetrate the mitochondrial matrix whereas pyruvate does. 3. Submitochondrial particles catalyse the ATP-driven reduction of NAD(+) by l-3-glycerophosphate but at a far lower rate than that seen when succinate is the electron donor. These particles do not have an energy-linked pyridine nucleotide transhydrogenase activity. 4. We conclude that the l-3-glycerophosphate-flavoprotein oxidoreductase is located on the outer surface of the inner membrane of the flight-muscle mitochondria.     

Control of the tricarboxylate cycle and its interactions with glycolysis during acetate utilization in rat heart

1. Transient and steady-state changes caused by acetate utilization were studied in perfused rat heart. The transient period occupied 6min and steady-state changes were followed in a further 6min of perfusion. 2. In control perfusions glucose oxidation accounted for 75% of oxygen utilization; the remaining 25% was assumed to represent oxidation of glyceride fatty acids. With acetate in the steady state, acetate oxidation accounted for 80% of oxygen utilization, which increased by 20%; glucose oxidation was almost totally suppressed. The rate of tricarboxylate-cycle turnover increased by 67% with acetate perfusion. The net yield of ATP in the steady state was not altered by acetate. 3. Acetate oxidation increased muscle concentrations of acetyl-CoA, citrate, isocitrate, 2-oxoglutarate, glutamate, alanine, AMP and glucose 6-phosphate, and lowered those of CoA and aspartate; the concentrations of pyruvate, ATP and ADP showed no detectable change. The times for maximum changes were 1min, acetyl-CoA, CoA, alanine and AMP; 6min, citrate, isocitrate, glutamate and aspartate; 2-4min, 2-oxoglutarate. Malate concentration fell in the first minute and rose to a value somewhat greater than in the control by 6min. There was a transient and rapid rise in glucose 6-phosphate concentration in the first minute superimposed on the slower rise over 6min. 4. Acetate perfusion decreased the output of lactate, the muscle concentration of lactate and the [lactate]/[pyruvate] ratio in perfusion medium and muscle in the first minute; these returned to control values by 6min. 5. During the first minute acetate decreased oxygen consumption and lowered the net yield of ATP by 30% without any significant change in muscle ATP or ADP concentrations. 6. The specific radioactivities of cycle metabolites were measured during and after a 1min pulse of [1-(14)C]acetate delivered in the first and twelfth minutes of acetate perfusion. A model based on the known flow rates and concentrations of cycle metabolites was analysed by computer simulation. The model, which assumed single pools of cycle metabolites, fitted the data well with the inclusion of an isotope-exchange reaction between isocitrate and 2-oxoglutarate+bicarbonate. The exchange was verified by perfusions with [(14)C]bicarbonate. There was no evidence for isotope exchange between citrate and acetyl-CoA or between 2-oxoglutarate and malate. There was rapid isotope equilibration between 2-oxoglutarate and glutamate, but relatively poor isotope equilibration between malate and aspartate. 7. It is concluded that the citrate synthase reaction is displaced from equilibrium in rat heart, that isocitrate dehydrogenase and aconitate hydratase may approximate to equilibrium, that alanine aminotransferase is close to equilibrium, but that aspartate transamination is slow for reasons that have yet to be investigated. 8. The slow rise in citrate concentration as compared with the rapid rise in that of acetyl-CoA is attributed to the slow generation of oxaloacetate by aspartate aminotransferase. 9. It is proposed that the tricarboxylate cycle may operate as two spans: acetyl-CoA-->2-oxoglutarate, controlled by citrate synthase, and 2-oxoglutarate-->oxaloacetate, controlled by 2-oxoglutarate dehydrogenase; a scheme for cycle control during acetate oxidation is outlined. The initiating factors are considered to be changes in acetyl-CoA, CoA and AMP concentrations brought about by acetyl-CoA synthetase. 10. Evidence is presented for a transient inhibition of phosphofructokinase during the first minute of acetate perfusion that was not due to a rise in whole-tissue citrate concentration. The probable importance of metabolite compartmentation is stressed.     

Unique features of flight muscles mitochondria of honey bees (Apis mellifera L.)

Honey bees Apis mellifera L. are one of the most studied insect species due to their economic importance. The interest in studying honey bees chiefly stems from the recent rapid decrease in their world population, which has become a problem of food security. Nevertheless, there are no systemic studies on the properties of the mitochondria of honey bee flight muscles. We conducted a research of the mitochondria of the flight muscles of A. mellifera L. The influence of various organic substrates on mitochondrial respiration in the presence or absence of adenosine diphosphate (ADP) was investigated. We demonstrated that pyruvate is the optimal substrate for the coupled respiration. A combination of pyruvate and glutamate is required for the maximal respiration rate. We also show that succinate oxidation does not support the oxidative phosphorylation and the generation of membrane potential. We also studied the production of reactive oxygen species by isolated mitochondria. The greatest production of H₂O₂ (as a percentage of the rate of oxygen consumed) in the absence of ADP was observed during the respiration supported by α‐glycerophosphate, malate, and a combination of malate with another NAD‐linked substrate. We showed that honey bee flight muscle mitochondria are unable to uptake Ca²⁺‐ions. We also show that bee mitochondria are able to oxidize the respiration substrates effectively at the temperature of 50°С compared to Bombus terrestris mitochondria, which were more adapted to lower temperatures.     

Changes in intramitochondrial adenine nucleotides in blowfly flight-muscle mitochondria

1. With freshly isolated blowfly mitochondria 38% of the intramitochondrial adenine nucleotide was present as AMP. 2. On incubation with oxidizable substrates the AMP and ADP concentrations fell and that of ATP rose; with pyruvate together with proline the ATP concentration reached its maximum value at 6min; with glycerol phosphate the phosphorylation of endogenous nucleotide was more rapid. 3. Addition of the uncoupling agent carbonyl cyanide phenylhydrazone caused a rapid fall of ATP and a parallel rise in ADP, then ADP was converted into AMP. 4. This was in contrast with rat liver mitochondria endogenous AMP concentrations, which were always lower than those of blowfly mitochondria and changed little under different metabolic conditions. 5. Evidence is presented that adenylate kinase (EC 2.7.4.3) has a dual distribution in blowfly mitochondria, a part being located in the matrix space and a part in the space between the outer and inner mitochondrial membranes, as in liver and other mitochondria. 6. The possible regulatory role of changing AMP concentrations in the mitochondrial matrix was investigated. Partially purified pyruvate carboxylase (EC 6.4.1.1) and citrate synthase (EC 4.1.3.7) were inhibited 30% by 2mm-AMP, whereas pyruvate dehydrogenase (EC 1.2.4.1) was unaffected. 7. AMP activated the NAD(+)-linked isocitrate dehydrogenase (EC 1.1.1.41) activity of blowfly mitochondria in the absence of ADP, but in the presence of ADP, AMP caused inhibition. 8. It is suggested that AMP may exert a controlling effect on the oxidative activity of blowfly mitochondria.     

Intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.     

The metabolism of glyoxylate by cell-free extracts of Pseudomonas sp

1. Extracts of Pseudomonas sp. grown on butane-2,3-diol oxidized glyoxylate to carbon dioxide, some of the glyoxylate being reduced to glycollate in the process. The oxidation of malate and isocitrate, but not the oxidation of pyruvate, can be coupled to the reduction of glyoxylate to glycollate by the extracts. 2. Extracts of cells grown on butane-2,3-diol decarboxylated oxaloacetate to pyruvate, which was then converted aerobically or anaerobically into lactate, acetyl-coenzyme A and carbon dioxide. The extracts could also convert pyruvate into alanine. However, pyruvate is not an intermediate in the metabolism of glyoxylate since no lactate or alanine could be detected in the reaction products and no labelled pyruvate could be obtained when extracts were incubated with [1-¹⁴C]glyoxylate. 3. The ¹⁴C was incorporated from [1-¹⁴C]glyoxylate by cell-free extracts into carbon dioxide, glycollate, glycine, glutamate and, in trace amounts, into malate, isocitrate and α-oxoglutarate. The ¹⁴C was initially incorporated into isocitrate at the same rate as into glycine. 4. The rate of glyoxylate utilization was increased by the addition of succinate, α-oxoglutarate or citrate, and in each case α-oxoglutarate became labelled. 5. The results are consistent with the suggestion that the carbon dioxide arises by the oxidation of glyoxylate via reactions catalysed respectively by isocitratase, isocitrate dehydrogenase and α-oxoglutarate dehydrogenase.     

Calcium inhibition of the NAD+-linked isocitrate dehydrogenase from blowfly flight muscle mitochondria

Free Ca2+ was shown to inhibit the NAD+-isocitrate dehydrogenase from blowfly flight muscle mitochondria. Inhibition by free Ca2+ concentrations of 40 microM or greater was found in the absence or presence of ADP and citrate, two known activators of the enzyme. Calcium decreased the affinity of the enzyme for its substrate, the magnesium DL-isocitrate chelate; no change in the apparent V of the reaction was observed. Calcium was inhibitory when activity was measured in the presence of fixed concentrations of magnesium DL-isocitrate chelate in the presence of several fixed concentrations of either free isocitrate3-, an activator, or free Mg2+, an inhibitor of the enzyme. That NAD+-isocitrate dehydrogenase from blowfly flight muscle mitochondria was not activated by micromolar free Ca2+ is consistent with the view that calcium does not play a role in regulating the flux through the tricarboxylate cycle in this species.     

The role of malic enzyme in the malate dependent biosynthesis of progesterone in the mitochondrial fraction of human term placenta

Mitochondria isolated from human term placenta were able to form citrate from malate as the only added substrate. While mitochondria were incubated in the presence of Mn2+ the citrate formation was stimulated significantly both by NAD+ and NADP+ and was inhibited by hydroxymalonate, arsenite, butylmalonate and rotenone. It is concluded that NAD(P)-linked malic enzyme is involved in the conversion of malate to citrate in these mitochondria. It has also been shown that the conversion of cholesterol to progesterone by human term placental mitochondria incubated in the presence of malate was stimulated by NAD+ and NADP+ and inhibited by arsenite and fluorocitrate. This suggests that the stimulation by malate of progesterone biosynthesis depends not only on the generation of NADPH by NAD(P)-linked malic enzyme, but also on NADPH formed during further metabolism of pyruvate to isocitrate which is in turn efficiently oxidized by NADP+-linked isocitrate dehydrogenase.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: pathways of C4 acid decarboxylation in bundle sheath cells of Urochloa panicoides

The mechanism of C4 acid decarboxylation was studied in bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C4 plant. Added malate was decarboxylated to give pyruvate and this activity was often increased by adding ADP. Added oxaloacetate or aspartate plus 2-oxoglutarate (which produce oxaloacetate via aspartate aminotransferase) gave little metabolic decarboxylation alone but with added ATP there was a rapid production of PEP. For this activity ADP could replace ATP but only when added in combination with malate. In addition, the inclusion of aspartate plus 2-oxoglutarate with malate plus ADP often increased the rate of pyruvate production from malate by more than twofold. Experiments with respiratory chain inhibitors showed that the malate-dependent stimulation of oxaloacetate decarboxylation (PEP production) was probably due to ATP generated during the oxidation of malate in mitochondria. We could provide no evidence that photophosphorylation could serve as an alternative source of ATP for the PEP carboxykinase reaction. We concluded that both PEP carboxykinase and mitochondrial NAD-malic enzyme contribute to C4 acid decarboxylation in these cells, with the required ATP being derived from oxidation-linked phosphorylation in mitochondria.     

L-lactate oxidation by skeletal muscle mitochondria

1. Mitochondria isolated from rat skeletal muscle possess lactate dehydrogenase which is involved in direct oxidation of L-lactate in the presence of external NAD. 2. L-lactate oxidation can be stimulated in a reversible manner by ADP. 3. Mitochondrial lactate oxidation is sensitive to oxamate-inhibitor of LDH, alpha-cyano-3-hydroxy-cinnamate-pyruvate translocase inhibitor and respiratory chain inhibitors (rotenone, antimycin A, KCN). 4. In the same conditions the mitochondria did not oxidize pyruvate in the absence of malate, whereas, oxidize pyruvate plus external NADH in an uncoupling manner.