Citrate Formation Research Articles

The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)+-dependent malic enzyme.

Little evidence has been available on the oxidative pathways of glutamine and glutamate, the major respiratory substrates of cancer cells. Glutamate formed from glutamine by phosphate-dependent glutaminase undergoes quantitative transamination by aerobic tumor mitochondria to yield aspartate. However, when malate is also added there is a pronounced decrease in aspartate production and a large formation of citrate and alanine, in both state 3 and 4 conditions. In contrast, addition of malate to normal rat heart, liver, or kidney mitochondria oxidizing glutamate causes a marked increase in aspartate production. Further analysis showed that extramitochondrial malate is oxidized almost quantitatively to pyruvate + CO2 by NAD(P)+-linked malic enzyme, present in the mitochondria of all tumors tested, but absent in heart, liver, and kidney mitochondria. On the other hand intramitochondrial malate generated from glutamate is oxidized quantitatively to oxalacetate by mitochondrial malate dehydrogenase of tumors. Acetyl-CoA derived from extramitochondrial malate via pyruvate and oxalacetate derived from glutamate via intramitochondrial malate are quantitatively converted into citrate, which is extruded. No evidence was found that malic enzyme of tumor mitochondria converts glutamate-derived malate into pyruvate as postulated in other reports. Possible mechanisms for the integration of mitochondrial malic enzyme and malate dehydrogenase activities in tumors are discussed.

Effect of treatment of rats with dexamethasone in vivo on gluconeogenesis and metabolite compartmentation in subsequently isolated hepatocytes

Hepatocytes prepared from rats treated with dexamethasone for 2 or 3h and maintained in the presence of 10 microM-dexamethasone in the preparation and incubation buffers showed significantly elevated rates of gluconeogenesis compared with those prepared from control animals. Dexamethasone treatment also increased the sensitivity of the cells to glucagon and the catecholamines. Analysis of the concentrations of metabolites in the gluconeogenic pathway indicated that dexamethasone decreased the intracellular concentration of pyruvate and increased those of phosphoenolpyruvate, acetyl-CoA and citrate, suggesting a stimulation of the reaction(s) converting pyruvate into phosphoenolpyruvate. This was substantiated by analysis of the pattern of metabolites found in the mitochondrial compartment after digitonin fractionation of the cells. Inclusion of 3-mercaptopicolinate in the incubation enhanced the effect of the hormone on the distribution of metabolites. Thus, in the absence of an effect of the steroid at the level of phosphoenolpyruvate carboxykinase or pyruvate kinase, dexamethasone treatment still increased the formation of malate, aspartate and citrate from pyruvate, indicating a stimulation in the intact cell of pyruvate carboxylase. It is suggested that the stimulation of pyruvate carboxylase is a result of a general activation of mitochondrial function, with an increase in the intramitochondrial concentrations of acetyl-CoA and ATP, a decrease in glutamate and an enhanced intramitochondrial [ATP]/[ADP] ratio.

Formation of coenzyme A from acetyl coenzyme A in pea leaf mitochondria: A requirement for oxaloacetate and the absence of hydrolysis

The release of coenzyme A-SH (CoA-SH) from acetyl CoA was measured in preparations of sonicated mithchondria prepared from Pisum sativum leaves on a discontinous Percoll gradient. Under our assay conditions, we found negligibsle releases of CoA-SH from acetyl CoA in the absence of a o-substrate. Co A-SH formation proceeded rapidly in the presence of oxaloacetate (OAA), indicating that the principal enzyme responsible for CoA-SH release is citrate synthase (EC 4.1.3.7.). OAA was consumed and citrate produced by this reaction. The apparent K m (acetyl CoA) was about 11–15 μM, and the apparent K m (OAA) was about 8–10 μM. We argue that in pea leaf mitochondria the great majority of the acetyl CoA produced by pyruvate oxidation will most likely enter into citrate formation and that the acetyl units will thereby pass into the tricarboxylic-acid cycle. Our data do not indicate any substantial hydrolytic release of CoA-SH from acetyl CoA. We therefore suggest that the acetyl-CoA hydrolase reported in spinach leaf mitochondria is not active in pea leaf mitochondria. Schemes have been proposed which invoke hydrolysis of mitochondrial acetyl CoA as a source of acetate for fatty acid synthesis in chloroplasts, but this type of scheme is evidently not valid for pea leaves.

The regulation of renal ammoniagenesis in the rat by extracellular factors. III. Effects of various fuels on in vitro ammoniagenesis

The addition of many oxidizable substrates to the medium of incubating rat renal slices decreases ammoniagenesis from glutamine and glutamate. Interestingly, lactate and β-hydroxybutyrate depress ammoniagenesis less in renal slices from acidotic rats compared with normal-control rats. In this study, the effects of an expanded panel of substrates on ammoniagenesis in kidney slices from control and acidotic rats were followed to discern patterns of inhibition. In addition to lactate and β-hydroxybutyrate, acetate, pyruvate, and perhaps acetoacetate caused relatively less depression of ammoniagenesis in acidotic slices. Citrate, succinate, fumarate, octanoate, and α-ketoglutarate decreased ammoniagenesis to the same extent or more in acidotic slices compared with that in normal-control slices. Glycerol had little effect on ammoniagenesis under either condition. From the substrates tested, it can be generalized that those outside the TCA cycle (with exception of octanoate) depress ammoniagenesis less during acidosis, while those in the TCA cycle depress ammoniagenesis equally or even more during acidosis. We hypothesize from the pattern of our results that changes in renal intermediary metabolism at or before citrate formation occur during acidosis and are important regulatory mechanisms for ammoniagenesis.

α-Ketoisocaproate stimulates malate-dependent pyruvate formation in rat heart mitochondria

In isolated rat heart mitochondria incubated with excess ADP and malate, α-ketoisocaproate (KIC) at 0.05–0.1 mM more than doubled the rate of citrate cycle activity (measured as citrate accumulation in the presence of fluorocitrate). Replacement of fluorocitrate with arsenite increased pyruvate formation stoichiometrically with the previous increase in citrate formation. No enhancement of pyruvate or citrate accumulation was seen when malate was omitted or when KIC was replaced with equimolar leucine. KIC (leucine) did not give rise to acetyl CoA, nor was pyruvate formed by way of oxaloacetate and phosphoenolpyruvate. These findings suggest that KIC regulated the formation of pyruvate from malate by acting as a positive modulator of mitochondrial malic enzyme.

Control of ketogenesis and fatty-acid synthesis at the mitochondrial branch-point for acetyl-CoA in the chick liver cell: effect of adenosine 3',5'-monophosphate.

Previous work has shown that dibutyryl adenosine 3′,5′‐monophosphate (Bt2cAMP) markedly activates ketogenesis from fatty acids released during the mobilization of cytoplasmic triacylglycerol‐rich vesicles in the chick liver cell. In the present investigation, activation of the formation of [14C]acetoacetate and 3‐[14C]hydroxybutyrate from [1‐14C]oleate by the cyclic nucleotide was shown to occur at two mitochondrial sites: (a) the l‐(–)‐carnitine‐dependent translocation of the fatty acid across the inner mitochondrial membrane and (b) the intramitochondrial metabolism of acetyl‐CoA.The hypothesis was tested that cAMP regulates ketogenesis and fatty acid synthesis by controlling acetyl‐CoA flux at the mitochondrial branch‐point leading into the 3‐hydroxy‐3‐methylglutaryl‐CoA cycle or to citrate formation. Bt2cAMP caused an immediate activation of acetoacetate formation and a concomitant inhibition of fatty acid synthesis from acetate. Evidence is presented that these effects are due to a cyclic‐nucleotide‐induced fall in the mitochondrial concentration of oxaloacetate. An assay for oxaloacetate, sufficiently sensitive to analyze extracts of cells in monolayer culture, was employed. With this assay, Bt, cAMP was shown to cause a > 90% decrease in cellular oxaloacetate concentration. The calculated level to which intramitochondrial oxaloacetate fell was below the Km for oxaloacetate in the citrate‐synthase‐catalyzed reaction and, therefore, was sufficient to curtail citrate formation.Lactate, a precursor of oxaloacetate and an antiketogenic agent, reversed the effects of Bt2cAMP. Under conditions where glucose was sole precursor of oxaloacetate and citrate, the activation of ketogenesis and the inhibition of fatty acid synthesis by Bt2cAMP were reversed by lactate. Lactate also reversed the Bt2cAMP‐induced decrease in oxaloacetate and citrate concentration. The fact that lactate enters the pathway between glucose and oxaloacetate indicated that the site of inhibition by Bt, cAMP lies above pyruvate in the glycolytic pathway. Based on these and previous results it appears that, by inhibiting glycolysis, cAMP exerts long‐range effects on two major pathways in the chick hepatocyte, ketogenesis and fatty acid synthesis.

Effect of inhibitors of RNA and protein synthesis on mitochondrial aspartate aminotransferase response to testosterone in rat ventral prostate.

The prostate gland is unusual in that it has a high content of citric acid compared with other soft tissues. The mechanism of citrate accumulation in prostate is unknown. One possibility is that aspartate aminotransferase (AAT) could supply an extra Krebs cycle source of oxaloacetate for citrate formation. Since testosterone regulates citrate production and secretion and strong transaminase activity exists in prostate, a relationship between AAT and citrate production could exist if testosterone also regulates AAT activity. To investigate this possibility, animals were castrated and given testosterone. After a total castration period of 72 hours, animals were sacrificed and mitochondrial AAT activity determined. Results revealed that castration decreased AAT activity and testosterone replacement stimulated activity. Neither castration nor testosterone affected malate dehydrogenase (MDH) activity. Both actinomycin D and cycloheximide blocked the testosterone stimulation of AAT activity. These studies indicate that testosterone may regulate AAT activity in rat ventral prostate. Furthermore, regulation may occur through testosterone stimulation of specific RNA and protein synthesis.

The energy-linked transhydrogenase in rat liver in relation to the reductive carboxylation of 2-oxoglutarate.

1 The energy-linked transhydrogenase reaction was studied in isolated mitochondria and in submitochondrial particles from rat liver. 2 The rate of the transhydrogenase reaction in intact mitochondria, estimated by measuring the rate of reduction of NADP+ with 3-hydroxybutyrate as hydrogen donor, was about 30 nmol · min−1· mg mitochondrial protein−1 at 25° C. This value was higher than that calculated from the rate of the energy-linked transhydrogenase measured in submitechondrial particles. From the ratio of the activities at 25° C and 37° C in submitochondrial particles and the rate at 25° C in intact mitochondria it was estimated that the rate of the transhydrogenase reaction in intact mitochondria at 37° C is about 80 nmol · min−1· mg protein−1. 3 In intact mitochondria the rate of reductive carboxylation of 2-oxoglutarate with 3-hydroxybutyrate as hydrogen donor was about 20 nmol · min−1· mg protein−1 at 37° C. The main product formed (> 95%) was citrate. 4 The concentration gradient of citrate across the mitochondrial membrane was the same during reductive carboxylation of 2-oxoglutarate as when citrate was added under non-flux conditions, indicating that transport of tricarboxylates does not limit citrate formation. 5 The activity of isocitrate dehydrogenase (NADP), measured in sonicated mitochondria in the direction of reductive carboxylation, was about 20 nmol · min−1· mg protein−1 at 37° C. 6 It is concluded that the rate of formation of citrate by reductive carboxylation of 2-oxoglutarate is limited by the activity of isocitrate dehydrogenase (NADP). However, the rate of citrate formation is sufficiently high to account for the transport of reducing equivalents needed for hydroxylation reactions in the extramitochondrial compartment in liver.

Comparison between developmental and senescent changes in enzyme activities linked to energy metabolism in rat heart

The developmental and senescent patterns of a number of heart enzyme activities linked to energy metabolism have been studied in rats aged between 4 days and 21 months. A morphometric study of mitochondrial volume fractions and numbers has been also carried out. Developmental changes result in a rise of most mitochondrial enzymes (NADP +-isocitrate dehydrogenase, malic enzyme, succinate dehydrogenase, citrate synthase) and mitochondrial volume fractions. Exceptions are NAD +-isocitarte dehydrogenase, which declines from 4 days onwards, and NAD +-malate dehydrogenase, which declines and then rises over the same period. Senescent changes follow two different trends. While pyruvate kinase and those mitochondrial enzymes lying between citrate formation and isocitrate oxidation (citrate synthase, NADP +- and NAD +-isocitrate dehydrogenases) decline to some degree, mitochondrial succinate dehydrogenase and NAD +-malate dehydrogenase activities increase over the same period. This could point towards a partial impairment of Krebs cycle function, and a reduced energy-producing capacity in the aged rat heart.

Infrared spectroscopic and chromatographic studies on uranium isotope effect in formation of uranyl acetate, citrate and fluoride complexes in aqueous solution

Abstract IR spectroscopic measurements were carried out on aqueous solutions of uranyl acetate, citrate, fluoride and perchlorate. The frequency shift for the asymmetric stretching vibration of the OUO bond was observed with different complexing anions. The reduced partition function ratios for uranium isotopes of uranyl complexes were calculated by using their frequencies. It was suggested that the lighter isotope 235 U should be accumulated more preferentially in uranyl complexes than in the hydrated uranyl ion. This result is in accord with that obtained by cation-exchange chromatography of solutions with compositions analogous to those for the IR spectral measurements.

Oxidation of NADH in Glyoxysomes by a Malate-Aspartate Shuttle

Glyoxysomes isolated from germinating castor bean endosperm accumulate NADH by beta-oxidation of fatty acids. By utilizing the glutamate: oxaloacetate aminotransferase and malate dehydrogenase present in glyoxysomes and mitochondria, reducing equivalents could be transferred between the organelles by a malate-aspartate shuttle. The addition of aspartate plus alpha-ketoglutarate to purified glyoxysomes brought about a rapid oxidation of accumulated NADH, and the oxidation was prevented by aminooxyacetate, an inhibitor of aminotransferase activity. Citrate synthetase activity in purified glyoxysomes could be coupled readily to glutamate: oxaloacetate aminotransferase activity as a source of oxaloacetate, but coupling to malate dehydrogenase and malate resulted in low rates of citrate formation. Glyoxysomes purified in sucrose or Percoll gradients were permeable to low molecular weight compounds. No evidence was obtained for specific transport mechanisms for the proposed shuttle intermediates. The results support a revised model of gluconeogenic metabolism incorporating a malate-aspartate shuttle in the glyoxysomal pathway.

A possible mechanism for the anti-ketogenic action of alanine in the rat.

1. The anti-ketogenic effect of alanine has been studied in normal starved and diabetic rats by infusing l-alanine for 90min in the presence of somatostatin (10mug/kg body wt. per h) to suppress endogenous insulin and glucagon secretion. 2. Infusion of alanine at 3mmol/kg body wt. per h caused a 70+/-11% decrease in [3-hydroxybutyrate] and a 58+/-9% decrease in [acetoacetate] in 48h-starved rats. [Glucose] and [lactate] increased, but [non-esterified fatty acid], [glycerol] and [3-hydroxybutyrate]/[acetoacetate] were unchanged. 3. Infusion of alanine at 1mmol/kg body wt. per h caused similar decreases in [ketone body] (3-hydroxybutyrate plus acetoacetate) in 24h-starved normal and diabetic rats, but no change in other blood metabolites. 4. Alanine [3mmol/kg body wt. per h] caused a 72+/-9% decrease in the rate of production of ketone bodies and a 57+/-8% decrease in disappearance rate as assessed by [3-(14)C]acetoacetate infusion. Metabolic clearance was unchanged, indicating that the primary effect of alanine was inhibition of hepatic ketogenesis. 5. Aspartate infusion at 6mmol/kg body wt. per h had similar effects on blood ketone-body concentrations in 48h-starved rats. 6. Alanine (3mmol/kg body wt. per h) caused marked increases in hepatic glutamate, aspartate, malate, lactate and citrate, phosphoenolpyruvate, 2-phosphoglycerate and glucose concentrations and highly significant decreases in [3-hydroxybutyrate] and [acetoacetate]. Calculated [oxaloacetate] was increased 75%. 7. Similar changes in hepatic [malate], [aspartate] and [ketone bodies] were found after infusion of 6mmol of aspartate/kg body wt. per h. 8. It is suggested that the anti-ketogenic effect of alanine is secondary to an increase in hepatic oxaloacetate and hence citrate formation with decreased availability of acetyl-CoA for ketogenesis. The reciprocal negative-feedback cycle of alanine and ketone bodies forms an important non-hormonal regulatory system.

Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.

Glucagon and N,(6)O(2)-dibutyryl cyclic adenosine 3',5'-cyclic monophosphate (Bt(2)cAMP) inhibit fatty acid synthesis from acetate by more than 90% and prevent citrate formation in chick hepatocytes metabolizing glucose. With substrates that enter glycolysis at or below triose-phosphates, e.g., fructose, lactate, or pyruvate, Bt(2)cAMP has no effect on the citrate level and its inhibitory effect on fatty acid synthesis is substantially reversed. Because acetyl-CoA carboxylase requires a tricarboxylic acid activator for activity, it is proposed that regulation of fatty acid synthesis by Bt(2)cAMP is due, in part, to changes in the citrate level. Reduced citrate formation appears to result from a cAMP-induced inhibition of glycolysis. Bt(2)cAMP inhibits (14)CO(2) production from [1-(14)C]-, [6-(14)C]-, and [U-(14)C]glucose and has little effect on (14)CO(2) formation from [1-(14)C]- or [2-(14)C]pyruvate or from [1-(14)C]fructose. [(14)C]Lactate formation from glucose is depressed 50% by Bt(2)cAMP. In the presence of an inhibitor of mitochondrial pyruvate transport lactate accumulation is enhanced, but continues to be lowered 50% by Bt(2)cAMP. The activity of phosphofructokinase is greatly decreased in Bt(2)cAMP-treated cells while the activities of pyruvate kinase and acetyl-CoA carboxylase are unaffected. It appears that decreased glycolytic flux and decreased citrate formation result from depressed phosphofructokinase activity. Fatty acid synthesis from [(14)C]acetate is partially inhibited by Bt(2)cAMP in the presence of fructose, lactate, and pyruvate despite a high citrate level. Incorporation of [(14)C]fructose, [(14)C]pyruvate, or [(14)C]lactate into fatty acids is similarly depressed by Bt(2)cAMP. Synthesis of cholesterol from [(14)C]acetate or [2-(14)C]pyruvate is unaffected by Bt(2)cAMP. These results implicate a second site of inhibition of fatty acid synthesis by Bt(2)cAMP that involves the utilization, but not the production, of cytoplasmic acetyl-CoA.-Clarke, S. D., P. A. Watkins, and M. D. Lane. Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.

An indirect method for studying the fine control of citric acid formation byAspergillus niger

During the main period of citric acid fermentation byAspernillus niger product formation is easily determinable from oxygen uptake and carbon dioxide output rates. This was applied in a study on the response of citrate formation to shifts of some environmental parameters of known importance during fermentation.

Role of AMP in regulation of the citric acid cycle in mitochondria from baker's yeast

The role of AMP in regulation of the citric acid cycle has been investigated in mitochondria prepared from mechanically disrupted baker's yeast ( Saccharomyces cerevisiae). Addition of citric acid cycle intermediates and the acetyl donors: pyruvate, acetaldehyde and acetate in micromolar concentrations caused discrete and reproducible oxygen uptake with a stoicheiometric relationship to substrate amount and metabolite formation, which allowed calculations of the extent of oxidation separately for acetyl donors and auxiliary substrate. AMP increased the rate of citrate oxidation in the mitochondrial suspensions to about the same level as found for oxidation of succinate and 2-oxoglutarate. The total oxygen consumption and malate formation agreed with oxidation through the citric acid cycle also for citrate, indicating insignificant contamination with peroxisomes and enzymes of the glyoxylate pathway. In the absence of AMP, addition of acetyl donor plus auxiliary substrate (malate for pyruvate and 2-oxoglutarate for acetaldehyde and acetate) led to citrate accumulation and an oxygen consumption which indicated that the auxiliary substrate had been utilized only once for citrate formation. Malate, succinate and citrate were unable to support acetate and acetaldehyde oxidation to the same extent as found with malate supported pyruvate oxidation. AMP increased the rate of acetyl donor oxidation, reduced citrate accumulation almost to zero and gave rise to complete oxidation of acetyl donors at molar acetyl donor/auxiliary substrate ratios up to 3. Complete oxidation of acetate was obtained also with citrate as auxiliary substrate, but not with malate or succinate. Half maximal stimulation of the rate of citrate and of acetate oxidation was acetate oxidation in the absence of AMP despite the concomitant oxidation 2-oxoglutarate. The reason for this failure is not clear. The low rate of citrate (isocitrate) oxidation might create conditions unfavourable for acetate activation e.g., by allowing side reactions to compete successfully for intermediates in substrate phosphorylation at the expense of reactions leading to acetate activation. In this case the AMP effects on acetate oxidation could be explained solely as a result of activation of the NAD-dependent isocitrate dehydrogenase. The results obtained in the present investigation are in general agreement with such a “one site” suggestion, but the results are on the other hand difficult to reconcile with the role of AMP in acetate activation considering that the activating system and isocitrate dehydrogenase are both localized at or inside the inner mitochondrial membrane. A more detailed study of acetate activation under the conditions employed in the present study seems therefore required.

Control of citrate and 2-oxoglutarate formation in Candida lipolytica mitochondria by adenine nucleotides

Candida lipolytica accumulates citrate from glucose mainly in the post- logarithmic growth phase. In yeast mitochondria prepared from cells in the logarithmic and stationary phases, citrate formation from pyruvate and malate was investigated. In the absence of added adenine nucleotides, the mitochondria from the stationary phase produced citrate, 2-oxoglutarate and a small amount of isocitrate with a 90% yield from consumed pyruvate on a molar base, while the yield was 58% with log-phase mitochondria. The effects of added adenine nucleotides on the formation of citrate and 2-oxoglutarate by stationary-phase mitochondria were studied and the following results were obtained: 1. 1. On addition of 0.1 mM ADP, a decreased citrate level and a 4-fold increase of 2-oxoglutarate formation were observed; 2. 2. Addition of 0.1 mM 2,4-dinitrophenol encouraged the production of both citrate and 2-oxoglutarate; 3. 3. AMP, similar to ADP, stimulated mitochondrial oxidation of citrate to 2-oxoglutarate, but AMP was effective at much lower concentrations than ADP; 4. 4. On addition of 0.3 mM ATP, citrate formation increased by about 50%. To explain the above results, isocitrate dehydrogenase (NAD +) (EC 1.1.1.41) in the mitochondrial extract was studied and it was found that it requires allosteric activation by AMP or ADP and that this stimulation is partly reversed by ATP. Concentrations of the adenine nucleotides in the cells of both growth phases were estimated. AMP content of the stationary-phase ccells was about 10% of that of log-phase cells. These results indicate that citrate formation by C. lipolytica is susceptibly controlled by adenine nucleotides, especially by AMP via the allosteric regulation of the activity of NAD +-isocitrate dehydrogenase.

Mechanism for acute control of fatty acid synthesis by glucagon and 3':5'-cyclic AMP in the liver cell.

Labeling experiments with chicken liver cell monolayers and suspensions show that glucagon and N6, O2-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP) block fatty acid synthesis from acetate without appreciably affecting cholesterogenesis from acetate or acylglyceride synthesis from palmitate. Neither acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] activity assayed in the presence of citrate nor fatty acid synthetase activity is decreased in extracts of cells treated with glucagon. However, the cytoplasmic concentration of citrate, a required allosteric activator of acetyl-CoA carboxylase, is depressed more than 90% by glucagon or dibutyrl cyclic AMP. Pyruvate or lactate largely prevents the inhibitory action of these effectors on fatty acid synthesis by causing a large increase in cytoplasmic citrate level. Thus, it appears that glucagon, acting via cyclic AMP, inhibits fatty acid synthesis by blocking the formation of citrate, an essential activator of acetyl-CoA carboxylase.

Rat lung mitochondrial transport: Factors affecting citrate formation

1. 1. Malate-dependent swelling of lung mitochondrial preparations in iso-osmotic ammonium citrate, and the inhibition of citrate formation by n-butylmalonate and benzene 1, 2, 3-tricarboxylate. suggest the presence of a malate-citrate exchange diffusion mechanism in lung mitochondria. 2. 2. d-Malate and 2-hydroxymalonate stimulated citrate formation from pyruvate and 2-oxoglutarate, suggesting that the presence of a suitable counter-ion for citrate efflux is a significant factor in the determination of extramitochondrial citrate availability. 3. 3. ADP-stimulated rates of pyruvate oxidation and citrate formation were inhibited by atractyloside. indicating the presence of the adenine nucleotide translocating system in lung tissue. 4. 4. Palmitoyl-CoA was found to inhibit citrate formation in lung mitochondrial preparations, presumably by an effect on the citrate translocator.

Sources of carbon for dicarboxylic acids in rat lung mitochondria

In view of previous work which demonstrated negligible pyruvate carboxylase activity, the present experiments were undertaken to investigate the potential roles of glutamate and the purine nucleotide cycle in replenishment of four-carbon units in rat lung mitochondria. 1. 1. Lung mitochondrial preparations were able to form citrate from 2-oxoglutarate, and, in the presence of 2-hydroxymalonate, the addition of L-glutamate resulted in net citrate formation. 2. 2. The rate of oxidation of [l- 14C] glutamate and [l- 14C] 2-oxoglutarate to 14CO 2 measured in these preparations was further increased in uncoupled mitochondria. 3. 3. Significant purine nucleotide cycle activity was observed in mitochondria-free lung tissue preparations. Additionally, significant net accumulation of ammonia, malate and fumarate was observed in the complete system; whereas when aspartate or GTP were omitted, no detectable fumarate and only low levels of ammonia and malate formation were observed. 4. 4. It is concluded that the limited ability to oxidize glutamate and the demonstration of purine nucleotide cycle activity provides at least 2 potential mechanisms for the maintenance of tricarboxylic acid cycle intermediates in lung mitochondria.

Enzymic generation of chiral acetates. A quantitative evaluation of their configurational assay.

1. R-Acetate was generated enzymically from R-acetate in the sequence acetate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. It was concluded that the formation of malate on malate synthase involves the operation of a normal isotopic effect combined with inversion of configuration. The malate synthase kH/k2H was determined as 3.7 +/- 0.5 by a method which yields results independently of the stereochemical purity of the chiral acetates used initially. 2. R-Acetate was also generated from R-acetate in the sequence acetate leads to citrate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. The conclusion is the same as given above, but refers to the formation of citrate on the re-synthase. 3. 2S,3R-[2-2H1,3-2H1,3H1]Malate and 2S,3S-[2-2H1,3-2H1]malate were prepared from 2S-[2,3-2H3]malate by treatment with fumarase in tritiated water and normal water, respectively. It was assumed that these malate specimens were pure with respect to chirality as generated by isotopic labelling. 4. These two malate specimens were partially converted (about 9%) to acetates in conditions where no racemization at the level of transiently formed oxaloacetate occurred. That no racemization took place was demonstrated experimentally. Oxidative enzymic hydrolysis of 2S,3R-[2-2H1,3-2H1,3H1]malate in normal water and of 2S,3S-[2-2H1,3-2H1]malate in tritiated water produced S-[2H1,3H1]acetate and R-[2H1,3H1]acetate, respectively. 5. The isolated R-[2H1,3H1]acetate and S-[2H1,3H1]acetate on configurational analysis yielded malates which in the presence of fumarase retained 79.7 +/- 0.7% and 20.3 +/- 0.9%, respectively, of their total tritium content. The symmetric deviation from the 50% value found with [3H1]acetate strengthens the conclusion that stereochemically pure chiral acetates were analyzed. The malate synthase kH/k2H was determined from the data of this study as 3.9 +/- 0.2. 6. The average of the values given under paragraphs 1 and 5 for the isotopic discrimination on malate synthase corresponds to kH/k2H=3.8 +/- 0.1. It was concluded that the configurational analysis of stereochemically pure R-[2H1,3H1]acetate and S-[2H1,3H1]acetate yields malates which in the presence of fumarase retain 79 +/- 2% and 21 +/- 2%, respectively, of their total tritium content. Hence, a deviation of 29 +/- 2% from the 50% value represents the actual amplitude of the configurational assay. 7. Outlines are given for an enzymic generation of chiral acetates in preparative scale.