Compartmentation of organic acids in corn roots I. Differential labeling of 2 malate pools

Bicarbonate-¹⁴C and acetate-³H were simultaneously provided to corn roots to give 2 isotopic forms of malate in the tissue, malate-¹⁴C produced by dark fixation reactions and malate-³H produced by reactions of the tricarboxylic acid cycle. Following a short pulse of exposure to the isotopes, the dissimilation of both isotopic forms of malic acid was followed. The rate of utilization of malate-³H was much faster than that of malate-¹⁴C.

These results are interpreted as showing that the malate produced from ¹⁴CO₂ is in a pool physically separated from that in the tricarboxylic acid cycle. The introduction of the 2 isotopes through distinct metabolic pathways produced the differential labeling of 2 distinct pools of malate.

     

Inhibition of Glutamine Transport in Rat Brain Mitochondria by Some Amino Acids and Tricarboxylic Acid Cycle Intermediates

Glutamine transport into rat brain synaptic and non-synaptic mitochondria has been monitored by the uptake of [³H]glutamine and by mitochondrial swelling. The concentration of glutamate in brain mitochondria is calculated to be high, 5–10 mM, indicating that phosphate activated glutaminase localized inside the mitochondria is likely to be dormant and the glutamine taken up not hydrolyzed. The uptake of [³H]glutamine is largely stereospecific. It is inhibited by glutamate, asparagine, aspartate, 2-oxoglutarate and succinate. Glutamate inhibits this uptake into synaptic and non-synaptic mitochondria by 95 and 85%, respectively. The inhibition by glutamate, asparagine, aspartate and succinate can be explained by binding to an inhibitory site whereas the inhibition by 2-oxoglutarate is counteracted by aminooxyacetic acid, which indicates that it is dependent on transamination. The glutamine-induced swelling, a measure of a very low affinity uptake, is inhibited by glutamate at a glutamine concentration of 100 mM, but this inhibition is abolished when the glutamine concentration is raised to 200 mM. This suggests that the very low affinity glutamine uptake is competitively inhibited by glutamate. Furthermore, glutamine-induced swelling is inhibited by 2-oxoglutarate, succinate and malate, similarly to that of the [³H]glutamine uptake. The properties of the mitochondrial glutamine transport are not identical with those of a recently purified renal glutamine carrier.     

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.     

Excess thymidine accelerates nascent replicon maturation without affecting the rate of DNA synthesis

The effects of different concentrations of exogenously supplied dThd on DNA replication were investigated in seedlings of Pisum sativum. Nascent DNA was labeled with either [³H]dThd or [³H]dAdo in the presence of 1·10^?6, 1·10^?5 or 1·10^?4 M unlabeled dThd. The rate of DNA synthesis was determined by measuring the kinetics of radioactivity incorporation into trichloroacetic acid-precipitable material and the size of the nascent molecules was investigated using alkaline sucrose gradients. The results obtained showed that high concentrations of exogenously supplied dThd accelerated the joining of completed nascent replicons without affecting the rate of DNA synthesis. This observation strengthens the hypothesis that the dTTP pool size is one of the factors controlling the timing of nascent replicon maturation.     

Utilization of exogenously supplied 14C-saccharose into primary metabolites and alkaloid production in Catharanthus roseus

Partitioning of exogenously supplied U-¹⁴C-saccharose into primary metabolic pool as sugars, amino acids, and organic acids was analyzed and simultaneous utilization for production of alkaloid by leaf, stem, and root in twigs and rooted plants of Catharanthus roseus grown in hydroponic culture medium was determined. Twigs revealed comparable distribution of total ¹⁴C label in leaf and stem. Stems contained significantly higher ¹⁴C label in sugar fraction and in alkaloids [47 kBq kg⁻¹(DM)] than leaf. In rooted plants, label in ¹⁴C in metabolic fractions in root such as ethanol-soluble, ethanol-insoluble, and chloroform-soluble fractions and in components such as sugars, amino acids, and organic acids were significantly higher than in stems and leaves. This was related with significantly higher content of ¹⁴C in alkaloids in stems and leaves. ¹⁴C contents in sugars, amino acids, and organic acids increased from leaf to stem and roots. Roots are the major accumulators of metabolites accompanied by higher biosynthetic utilization for alkaloid accumulation.     

Metabolism of propionate by sheep liver: Pathway of propionate metabolism in aged homogenate and mitochondria

Experiments were conducted with aged nuclear-free homogenate of sheep liver and aged mitochondria in an attempt to measure both the extent of oxidation of propionate and the distribution of label from [2-¹⁴C]propionate in the products. With nuclear-free homogenate, propionate was 44% oxidized with the accumulation of succinate, fumarate, malate and some citrate. Recovery of ¹⁴C in these intermediates and respiratory carbon dioxide was only 33%, but additional label was detected in endogenous glutamate and aspartate. With washed mitochondria 30% oxidation of metabolized propionate occurred, and proportionately more citrate and malate accumulated. Recovery of ¹⁴C in dicarboxylic acids, citrate, α-oxoglutarate, glutamate, aspartate and respiratory carbon dioxide was 91%. The specific activities of the products and the distribution of label in the carbon atoms of the dicarboxylic acids were consistent with the operation solely of the methylmalonate pathway together with limited oxidation of the succinate formed by the tricarboxylic acid cycle via pyruvate. In a final experiment with mitochondria the label consumed from [2-¹⁴C]propionate was entirely recovered in the intermediates of the tricarboxylic acid cycle, glutamate, aspartate, methylmalonate and respiratory carbon dioxide.     

Regulation of succinate oxidation by NAD+ in mitochondria purified from potato tubers

NAD⁺ supplied to purified Solanum tuberosum mitochondria caused progressive inhibition of succinate oxidation in State 3. This inhibition was especially pronounced at alkaline pH and at low succinate concentrations. Glutamate counteracted the inhibition. NAD⁺ promoted oxaloacetate accumulation in State 3; supplied oxaloacetate inhibited O₂ uptake in the presence of succinate much more severely in State 3 than in State 4. NAD reduction linked to succinate oxidation by ATP-dependent reverse electron transport was likewise inhibited by oxaloacetate. We conclude that NAD⁺-induced inhibition of succinate oxidation is due to an inhibition of succinate dehydrogenase resulting from increased accumulation of oxaloacetate generated from malate oxidation via malate dehydrogenase. The results are discussed in the context of the known regulatory characteristics of plant succinate dehydrogenase.     

Compartmentation of Organic Acids in Corn Roots II. The Cytoplasmic Pool of Malic Acid

The major conclusion drawn was that malate generated in corn roots during a 15-minute period of CO₂ fixation and malate introduced into the tissue during a similar period from the bathing medium share a common extramitochondrial compartment, the cytoplasmic pool. The utilization of these 2 forms of malate is normally much slower than that of malate generated in the mitochondria by the tricarboxylic acid cycle. By lowering the pH of the medium or treating the tissue with malonate or 2,4-dinitrophenol, similar increases in the rates of utilization of both forms of cytoplasmic malate were brought about. Changes in (A) the demand for acetyl acceptors in the mitochondria and (B) mitochondrial permeability were invoked to account for the increased utilization of the cytoplasmic malate under the various experimental treatments.     

Glutamic Acid metabolism and the photorespiratory nitrogen cycle in wheat leaves: metabolic consequences of elevated ammonia concentrations and of blocking ammonia assimilation

The effects of methionine sulfoximine and ammonium chloride on [¹⁴C] glutamate metabolism in excised leaves of Triticum aestivum were investigated. Glutamine was the principal product derived from [U¹⁴C]glutamate in the light and in the absence of inhibitor or NH₄Cl. Other amino acids, organic acids, sugars, sugar phosphates, and CO₂ became slightly radioactive. Ammonium chloride (10 mm) increased formation of [¹⁴C] glutamine, aspartate, citrate, and malate but decreased incorporation into 2-oxoglutarate, alanine, and ¹⁴CO₂. Methionine sulfoximine (1 mm) suppressed glutamine synthesis, caused NH₃ to accumulate, increased metabolism of the added radioactive glutamate, decreased tissue levels of glutamate, and decreased incorporation of radioactivity into other amino acids. Methionine sulfoximine also caused most of the ¹⁴C from [U-¹⁴C]glutamate to be incorporated into malate and succinate, whereas most of the ¹⁴C from [1-¹⁴C]glutamate was metabolized to CO₂ and sugar phosphates. Thus, formation of radioactive organic acids in the presence of methionine sulfoximine does not take place indirectly through “dark” fixation of CO₂ released by degradation of glutamate when ammonia assimilation is blocked. When illuminated leaves supplied with [U-¹⁴C] glutamate without inhibitor or NH₄Cl were transferred to darkness, there was increased metabolism of the glutamate to glutamine, aspartate, succinate, malate, and ¹⁴CO₂. Darkening had little effect on the labeling pattern in leaves treated with methionine sulfoximine.     

Simultaneous measurement of oxidative phosphorylation and adenylate kinase in plant mitochondria

An assay system capable of simultaneously measuring ATP, ADP, and AMP concentrations was used for the measurement of oxidative phosphorylation and adenylate kinase (5′-ATP:5′-AMP phosphotransferase) activities in mitochondria which were isolated from etiolated corn, soybean, or cucumber seedlings. Data obtained by this system was correlated with colorimetric P_i uptake and spectrophotometric NADH oxidation measurements. Adenylate kinase was active in both phosphorylating and nonphosphorylating mitochondria. Studies using NaCN, 2,4-dinitrophenol, atractyloside, and 2′-AMP as inhibitors indicated that exogenously supplied [¹⁴C]AMP was converted to [¹⁴C]ADP either by NADH-linked phosphorylation or by translocation and transphosphorylation from intramitochondrial nucleotides.     

Involvement of glutamate in the respiratory metabolism of Bradyrhizobium japonicum bacteroids.

Bradyrhizobium japonicum bacteroids were isolated anaerobically and supplied with 14C-labeled succinate, malate, aspartate, or glutamate for periods of up to 60 min in the presence of myoglobin to control the O2 concentration. Succinate and malate were absorbed about twice as rapidly as glutamate and aspartate. Conversion of substrate to CO2 was most rapid for malate, followed by succinate, glutamate, and aspartate. When CO2 production was expressed as a proportion of total carbon taken up, malate was still the most rapidly respired substrate, with 68% of the label absorbed converted to CO2. The comparable values for succinate, glutamate, and aspartate were 37, 50, and 38%, respectively. Considering the fate of labeled substrate not respired, greater than 95% of absorbed glutamate remained as glutamate in the bacteroids. In contrast, from 39 to 66% of the absorbed succinate, malate, or aspartate was converted to glutamate. An increase in the rate of CO2 formation from labeled substrates after 20 min appeared to coincide with a maximum accumulation of label in glutamate. The results indicate the presence of a substantial glutamate pool in bacteroids and the involvement of glutamate in the respiratory metabolism of bacteroids.     

Indoleacetic Acid synthesis in soybean cotyledon callus tissue

Growth of an auxin-requiring soybean cotyledon callus tissue (Glycine max L., Merr. var. Acme) was promoted by tryptophan, tryptamine, indole, indoleacetamide and, to a very slight degree, anthranilic acid. When tryptophan-3-¹⁴C was supplied in the growth medium, labeled indoleacetic acid (IAA) was found in both the tissue and the medium. Medium, from which the cells had been removed, was also found to convert labeled tryptophan to IAA. Soybean callus contained 0.044 μmole/g free tryptophan, but this is apparently not available for conversion to IAA. These results suggest that while exogenously supplied trytophan could elevate a specific internal pool where IAA synthesis occurs some of the growth on a tryptophan medium can be accounted for by external conversion.     

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

METABOLIC COMPARTMENTATION IN THE BRAIN: METABOLISM OF A TRICARBOXYLIC ACID CYCLE INTERMEDIATE, [1,4-14C] SUCCINATE, AFTER INTRACEREBRAL ADMINISTRATION

Abstract— The metabolism of a tricarboxylic acid cycle (cycle) intermediate, [1.4-'¹⁴C]succinate, was studied in the brain at 2 20 min after intracerebral injection. The oxidation of [¹⁴C]succinate was rapid, as shown by the incorporation of ¹⁴C into cycle amino acids which accounted for about 30 per cent and 70 per cent of the tissue -“Cat 2 and 10 min respectively. During the whole experimental period the specific radioactivity of glutamine was about three times higher than that of glutamate. Thus exogenous [¹⁴C]succinate elicited signs of metabolic compartmentation similar to those seen after the administration of short chain fatty acids or amino acids. A computer programme, based on data obtained previously on the metabolic compartmentation of acetate and of glucose in the brain, was used to simulate the kinetics of labelling of cycle amino acids after an input of [1.4-¹⁴C]succinate. The correspondence of the simulated data with the experimental results was good in the first 10 min after injection, although the deviations were significant at later time points. Incorporation of ¹⁴C into GABA was very low (< 1 per cent of the amino acid -¹⁴C) after the injection of [1.4-¹⁴C]succinate. Further, labelled GABA formation was not detected in the decapitated rat brain labelled in vivo with [1.4-¹⁴C]succinate 2 min beforehand. Since the oxidation of [l,4-¹⁴C]succinate via the cycle yields unlabellcd GABA. whereas the reversal of the reactions in the GABA bypath may introduce ¹⁴C from succinate into the GABA pool, the results indicate that this reversal is negligible even under the most favourable conditions, i.e. post mortem when both the NADH/NAD⁺ ratios and [¹⁴C]succinate concentrations arc high. The observations are therefore consistent with the view that glutamate is the predominant and probably the only source of GABA carbon in the brain both in vivo and post mortem.     

BIOSYNTHESIS IN ISOLATED ACETABULARIA CHLOROPLASTS : I. Protein Amino Acids

The ability of chloroplasts isolated from Acetabulana mediterranea to synthesize the protein amino acids has been investigated. When this chloroplast isolate was presented with ¹⁴CO₂ for periods of 6–8 hr, tracer was found in essentially all amino acid species of their hydrolyzed protein Phenylalanine labeling was not detected, probably due to technical problems, and hydroxyproline labeling was not tested for The incorporation of ¹⁴CO₂ into the amino acids is driven by light and, as indicated by the amount of radioactivity lost during ninhydrin decarboxylation on the chromatograms, the amino acids appear to be uniformly labeled. The amino acid labeling pattern of the isolate is similar to that found in plastids labeled with ¹⁴CO₂ in vivo. The chloroplast isolate did not utilize detectable amounts of externally supplied amino acids in light or, with added adenosine triphosphate (ATP), in darkness. It is concluded that these chloroplasts are a tight cytoplasmic compartment that is independent in supplying the amino acids used for its own protein synthesis. These results are discussed in terms of the role of contaminants in the observed synthesis, the "normalcy" of Acetabularia chloroplasts, the synthetic pathways for amino acids in plastids, and the implications of these observations for cell compartmentation and chloroplast autonomy.     

Synthesis of proteins and nucleic acids by the pea aphid Acyrthosiphon pisum (aphididae) in the absence of a full complement of dietary amino acids

Protein, nucleic acids, and nucleotide syntheses were studied in pea aphids, Acyrthosiphon pisum (Harris), by feeding them labeled ¹⁴C-amino acids and [5-³H]-orotic acid in sucrose. It was demonstrated that in the absence of dietary essential amino acids, aphids were capable of synthesizing nucleic acids, nucleotides, and proteins when provided with a single dietary amino acid in sucrose. It is suggested that other required amino acids were possibly supplied by the symbionts present in the pea aphid and/or were obtained from the amino acid pool in the hemolymph or glucose, one of the end products of sucrose digestion. Of the various amino acids tested, synthesis of measurable amounts of protein or other compounds occurred when alanine, aspartic acid, glutamic acid, glycine, proline, or serine were provided, but no synthesis occurred with cysteine.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence for the operation of a reductive tricarboxylic acid cycle in growing cells

Chlorobium limicola was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C propionate and the incorporation of ¹⁴C into alanine ( intracellular pyruvate), aspartate ( oxaloacetate), and glutamate ( -ketoglutarate) was studied in long term labeling experiments. During growth in presence of propionate 30% of the cell carbon were derived from propionate and 70% from CO₂. Propionate was not oxidized to CO₂.All three amino acids were found to be labeled. The labeling patterns indicate that propionate was assimilated via propionyl CoA, methylmalonyl CoA and succinyl CoA. When 1-¹⁴C propionate was the labeled precursor no radioactivity was found in the carboxyl group(s) of alanine, aspartate and glutamate, excluding the incorporation of propionate into the amino acids via succinate oxidation to fumarate. With 1-¹⁴C propionate preferentially aspartate (C-3) and glutamate (C-2) became labeled, with 2-¹⁴C propionate alanine (C-3) and glutamate (C-4). These findings indicate that propionate was incorporated into the amino acids via succinyl CoA, -ketoglutarate, isocitrate, and citrate, followed by a si-type cleavage of citrate to oxaloacetate and acetyl CoA (or acetate). Similar experiments with U-¹⁴C acetate confirm these conclusions. Thus, all reactions of the proposed reductive tricarboxylic acid cycle could be demonstrated in autotrophically growing cells.     

Succinate metabolism related to lipid synthesis in the housefly Musca domestica L

The metabolism of succinate was examined in the housefly Musca domestica L. The labeled carbons from [2,3-¹⁴C]succinate were readily incorporated into cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,4-¹⁴C]succinate was not incorporated into either fraction. Examination of the incorporation of [2,3-¹⁴C]succinate, [1-¹⁴C]acetate, and [U-¹⁴C]proline into hydrocarbon by radio-gas-liquid chromatography showed that each substrate gave a similar labeling pattern, which suggested that succinate and proline were converted to acetyl-CoA prior to incorporation into hydrocarbons. Carbon-13 nuclear magnetic resonance showed that the labeled carbons from [2,3-¹³C]succinate enriched carbons 1, 2, and 3 of hydrocarbons with carbon-carbon coupling showing that carbons 2 and 3 of succinate were incorporated as an intact unit. Radio-high-performance liquid chromatographic analysis of [2,3-¹⁴C]succinate metabolism by mitochondrial preparations showed that in addition to labeling fumarate, malate, and citrate, considerable radioactivity was also present in the acetate fraction. The data show that succinate was not converted to methylmalonate and did not label hydrocarbon via a methylmalonyl derivative. Malic enzyme was assayed in sonicated mitochondria prepared from the abdomens and thoraces of 1- and 4-day-old insects; higher activity was obtained with NAD⁺ in mitochondria prepared from thoraces, whereas NADP⁺ gave higher activity with abdomen preparations. These data document the metabolism of succinate to acetyl-CoA and not to a methylmalonyl unit prior to incorporation into lipid in the housefly and establish the role of the malic enzyme in this process.     

Dark Respiration during Photosynthesis in Wheat Leaf Slices

The metabolism of [¹⁴C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-¹⁴C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO₂. An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after ¹⁴CO₂ production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of ¹⁴CO₂ over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO₂ as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO₂ was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the ¹⁴CO₂ release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.     

Role of aspartate aminotransferase and mitochondrial dicarboxylate transport for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons

Evoked release of glutamate and aspartate from cultured cerebellar granule cells was studied after preincubation of the cells in tissue culture medium with glucose (6.5 mM), glutamine (1.0 mM),d[³H] aspartate and in some cases aminooxyacetate (5.0 mM) or phenylsuccinate (5.0 mM). The release of endogenous amino acids and ofd-[³H] aspartate was measured under physiological and depolarizing (56 mM KCl) conditions both in the presence and absence of calcium (1.0 mM), glutamine (1.0 mM), aminooxyacetate (5.0 mM) and phenylsuccinate (5.0 mM). The cellular content of glutamate and aspartate was also determined. Of the endogenous amino acids only glutamate was released in a transmitter fashion and newly synthesized glutamate was released preferentially to exogenously suppliedd-[³H] aspartate, a marker for exogenous glutamate. Evoked release of endogenous glutamate was reduced or completely abolished by respectively, aminooxyacetate and phenylsuccinate. In contrast, the release ofd-[³H] aspartate was increased reflecting an unaffected release of exogenous glutamate and an increased psuedospecific radioactivity of the glutamate transmitter pool. Since aminooxyacetate and phenylsuccinate inhibit respectively aspartate aminotransferase and mitochondrial keto-dicarboxylic acid transport it is concluded that replenishment of the glutamate transmitter pool from glutamine, formed in the mitochondrial compartment by the action of glutaminase requires the simultaneous operation of mitochondrial keto-dicarboxylic acid transport and aspartate aminotransferase which is localized both intra- and extra-mitochondrially. The purpose of the latter enzyme apparently is to catalyze both intra- and extra-mitochondrial transamination of -ketoglutarate which is formed intramitochondrially from the glutamate carbon skeleton and transferred across the mitochondrial membrane to the cytosol where transmitter glutamate is formed. This cytoplasmic origin of transmitter glutamate is in aggreement with the finding thatd-[³H] aspartate readily labels the transmitter pool even when synthesis of endogenous transmitter is impaired in the presence of AOAA or phenylsuccinate.Special issue dedicated to Dr Elling Kvamme