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.     

The Effect of Light on the Tricarboxylic Acid Cycle in Green Leaves: III. A Comparison between Some C(3) and C(4) Plants

The chlorophyll-based specific activity of cytochrome oxidase and three exclusively mitochondrial enzymes of the tricarboxylic acid cycle showed little variation between leaves of C₃ and C₄ plants or between mesophyll and bundle sheath cells of Atriplex spongiosa and Sorghum bicolor. However, a large, light-dependent transfer of label from intermediates of the tricarboxylic acid cycle to photosynthetic products was a feature of leaves of C₄ plants. This light-dependent transfer of label was barely detectable in leaves of C₃ plants and in leaves of F₁ and F₃ hybrids of Atriplex rosea (C₄) and Atriplex patula spp hastata (C₃). The light-dependent transfer of label to photosynthetic products in leaves of C₄ plants was inhibited by the tricarboxylic acid cycle inhibitors malonate and fluoroacetate. The requirement for continued tricarboxylic acid cycle activity was also indicated in experiments with specifically labeled succinate-¹⁴C. These experiments, together with the distribution of ¹⁴C in glucose prepared from sucrose-¹⁴C formed during the metabolism of succinate-2,3-¹⁴C, confirmed that the photosynthetic metabolism of malate and aspartate derived from the tricarboxylic acid cycle, and not the refixation of respiratory CO₂, was the main path of carbon from the cycle to photosynthesis.     

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.     

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.     

Anaerobic incorporation of radioactivity from 2,3-14C-succinic acid into citric acid cycle intermediates and related compounds in the sea musselMytilus edulis L.

Summary Sea mussels were exposed to nitrogen for various periods (0, 1, 3 and 6 days) and subsequently injected with 2,3-¹⁴C-succinic acid. After 2.5 h anaerobic incubation concentrations of succinate, some amino acids and volatile fatty acids were determined as well as the distribution of radioactivity.Conversion of the precursor decreased from 80 to 40%, due to increased dilution with endogenous succinate, accumulated during the anaerobic preincubation period.More than 80% of the activity of the converted 2,3-¹⁴C-succinic acid was incorporated into malate, aspartate, glutamate, alanine and propionate. This indicates that succinate is not only an end product of anaerobic glycogen breakdown, but remains an active intermediate of the tricarboxylic acid cycle, which can still operate under anaerobic conditions.Concentration and radioactivity of propionate were markedly increased after prolonged anoxia, which gives evidence that succinate is actively converted to propionate during anaerobiosis.Observed accumulation of glutamate during anoxia is explained by incomplete oxidation of pyruvate, which leaves the tricarboxylic acid cycle at the stage of 2-ketoglutarate.     

Effects of Light and Inhibitors on Glutamate Metabolism in Leaf Discs of Vicia faba L: Sources of ATP for Glutamine Synthesis and Photoregulation of Tricarboxylic Acid Cycle Metabolism

Metabolism of [¹⁴C]glutamate was studied in leaf discs of Vicia faba L. in light and in darkness. In white light glutamine was the main labeled product. In the dark label was principally in compounds closely associated with tricarboxylic acid cycle metabolism, predominantly aspartate. Entry of label from glutamate into tricarboxylic acid metabolism appeared to be at least partially by decarboxylation of glutamate to γ-amino butyric acid, followed by conversion to succinate. 3-(3,4-dichlorophenyl)-1, 1-Dimethylurea inhibited light-enhanced synthesis of glutamine and caused reversion toward the dark pattern of metabolism. Methionine sulfoximine severely inhibited glutamine synthesis and caused accumulation of labeled malate.     

In VivoC NMR Study of the Bidirectional Reactions of the Wood–Werkman Cycle and around the Pyruvate Node in Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici

This study used in vivo¹³C NMR spectroscopy to directly examine bidirectional reactions of the Wood–Werkman cycle involved in central carbon metabolic pathways of dairy propionibacteria during pyruvate catabolism. The flow of [2-¹³C]pyruvate label was monitored on living cell suspensions of Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici under acidic conditions. P. shermanii and P. acidipropionici cells consumed pyruvate at apparent initial rates of 161 and 39 μmol min⁻¹ g⁻¹ (cell dry weight), respectively. The bidirectionality of reactions in the first part of the Wood–Werkman cycle was evident from the formation of intermediates such as [3-¹³C]pyruvate and [3-¹³C]malate and of products like [2-¹³C]acetate from [2-¹³C]pyruvate. For the first time alanine labeled on C2 and C3 and aspartate labeled on C2 and C3 were observed during [2-¹³C]pyruvate metabolism by propionibacteria. The kinetics of aspartate isotopic enrichment was evidence for its production from oxaloacetate via aspartate aminotransferase. Activities of a partial tricarboxylic acid pathway, acetate synthesis, succinate synthesis, gluconeogenesis, aspartate synthesis, and alanine synthesis pathways were evident from the experimental results.     

Carbon Assimilation in Photoheterotrophic Cells of Peanut (Arachis hypogaea L.) Grown in Still Nutrient Medium

The relative transport of photosynthetic and dark carboxylation products in photoheterotrophic cells of Arachis hypogaea L. var. TMV-3 at varied phases of growth were determined. Despite the presence of an equally competent photosynthetic apparatus as determined from ¹⁴CO₂ incorporation rates in the dark and light, pulse-chase experiments revealed little or no change in the radioactivity of the C₃ intermediates but rapid disappearance of label from the dark carbon assimilates (malate and other tricarboxylic acid cycle intermediates) with a simultaneous increase in the aminoacid pool in early log-phase (10 days old) cells. However, significant flow of carbon through the photosynthetic intermediates resulting in the accumulation of sugars occurred in the late log-phase (34 days old) cells. Limitation of exogenous sugar in the nutrient milieu and depletion of reserve carbohydrates stored in starch of the chloroplasts of the cells were considered as the decisive factors in promoting transport of C₃ cycle intermediates through the reductive pentose phosphate pathway in photoheterotrophic cells. The observed drain of radioactivity even from the small amounts of tricarboxylic acid cycle intermediates synthesized during photosynthesis into glutamate indicated that the transport of carbon through the nonautotrophic pathway is not controlled by these factors.     

Intermediary metabolism of carbon compounds by nitrifying bacteria

Summary The assimilation of¹⁴CO₂ and [2-¹⁴C] acetate, [3-¹⁴C] pyruvate, [5-¹⁴C] -ketoglutarate, [2,3-¹⁴C] succinate, [U-¹⁴C] glutamate and [U-¹⁴C] aspartate was followed in cell suspensions ofNitrosomonas europaea andNitrobacter agilis respectively. There was appreciable incorporation of these substrates even without adding the inorganic nitrogen compounds that are oxidized by these bacteria yielding ATP. In the soluble amino acid fraction most of¹⁴C label was recovered in glutamate while in the protein amino acids a more uniform distribution was found. Acetate was rapidly incorporated to a high level in both nitrifying bacteria while inNitrobacter there was a relatively lower uptake of the other substrates especially succinate. High levels of the NAD malate dehydrogenase and NADP isocitrate dehydrogenase were measured but no significant amounts of the other tricarboxylic acid cycle enzymes or NADH oxidase were found. Glutamate decarboxylase was detected in both organisms and the transferase assay for glutamine synthetase indicated a 30-fold higher activity for this enzyme inNitrobacter. The amino acid composition of the water soluble fraction was determined in both bacteria.     

Metabolic physiology of alcohol degradation and adaptation in Drosophila larvae as studied by means of carbon-13 nuclear magnetic resonance spectroscopy

Alcohol dehydrogenase-mediated degradation of [2-¹³C]ethanol was followed in third instar larvae of Drosophila by means of ¹³C NMR. The tricarboxylic acid (TCA) cycle intermediates, citrate-C(2),4 and succinate-C2,3; the amino acids, glutamate-C4,3,2, glutamine-C4,3,2, proline-C4, alanine-C2,3 and the carbon nuclei of the glucosyl units of the disaccharide, α,α-trehalose, were intensely labeled in perchloric acid extracts of whole larvae. A model of the intermediary metabolism of ethanol degradation in larvae was formulated from these observations. The C2 atom of ethanol enters the mitochondrial TCA cycle as C2-acetyl-CoA and is converted into the TCA cycle intermediates. The TCA cycle intermediate 2-oxoglutarate(-C4) apparently is readily converted into glutamate(-C4) and subsequently to glutamine(-C4) and proline(-C4). Dietary ethanol is also a substrate for trehalose synthesis. This may occur by an exchange of malate(-C2,3) between its mitochondrial and cytosolic pools. Part of the cytosolic malate(-C2,3) may be diverted into pyruvate then generating alanine(-C2,3) as another product. The other part may be converted into glucose and subsequently into α,α-trehalose by the gluconeogenic pathway. ¹³C natural abundance signals of stored fatty acids and glycerol were readily detectable in chloroform extracts of control larvae. De novo synthesis of fatty acids from labeled ethanol also occurred after a lag period. Our findings show the coordinated nature of metabolic pathways, and we point to its consequences in understanding the dynamics in evolutionary processes.     

CO2-Fixierung und Stofftransport in benthischen marinen Algen

Summary When discs punched out of the median part of the phylloid of Laminaria saccharina Lamour. were exposed to H¹⁴CO₃ ^- in the light for periods of 10 sec to 10 min, ¹⁴C was rapidly incorporated into various photosynthetic products. As compared with dark fixation, ¹⁴C-photosynthesis increased exponentially during the first 60 sec of incubation in H¹⁴CO₃ ^-. Fixation rates were found to be 76 mol CO₂·dm^-2·h^-1 or 100 mol CO₂·mg^-1 chlorophyll a·h^-1. Eighty-five per cent of the total ¹⁴C assimilated after 10 sec was fixed in phosphoglycerate and in the sugar monophosphates, 2% in the sugar diphosphates, and only 3.5% in malate and aspartate. While the radioactivity of malate and aspartate only rose to a constant level, the percentage of the total ¹⁴C in phosphoglycerate and-to a lower extent-that in the sugar monophosphates rapidly decreased with the duration of light exposure. Simultaneously, mannitol and glycine+serine became labelled with 43% and 32% respectively of the total ¹⁴C after 10 min light fixation. In the dark, the percentage of the total ¹⁴C in malate decreased with the time of H¹⁴CO^2--incubation, while there was a remarkable increase in radioactivity of aspartate and glutamate. Within 60 min darkness no labelling of mannitol was found.From the present results it is concluded that the photosynthetic carbon cycle first described by Bassham and Calvin operates in Laminaria saccharina.

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Auszug aus einer Diplomarbeit.     

d-Glycerate Transport by the Pea Chloroplast Glycolate Carrier : Studies on [1-C]d-Glycerate Uptake and d-Glycerate Dependent O(2) Evolution

Rapid direct conversion of exogenously supplied [¹⁴C]aspartate to [¹⁴C] asparagine and to tricarboxylic cycle acids was observed in alfalfa (Medicago sativa L.) nodules. Aspartate aminotransferase activity readily converted carbon from exogenously applied [¹⁴C]aspartate into the tricarboxylic acid cycle with subsequent conversion to the organic acids malate, succinate, and fumarate. Aminooxyacetate, an inhibitor of aminotransferase activity, reduced the flow of carbon from [¹⁴C]aspartate into tricarboxylic cycle acids and decreased ¹⁴CO₂ evolution by 99%. Concurrently, maximum conversion of aspartate to asparagine was observed in aminooxyacetate treated nodules (30 nanomoles asparagine per gram fresh weight per hour. Metabolism of [¹⁴C]aspartate and distribution of nodulefixed ¹⁴CO₂ suggest that two pools of aspartate occur in alfalfa nodules: (a) one involved in asparagine biosynthesis, and (b) another supplying a malate/aspartate shuttle. Conversion of [¹⁴C]aspartate to [¹⁴C]asparagine was not inhibited by methionine sulfoximine, a glutamine synthetase inhibitor, or azaserine, a glutmate synthetase, inhibitor. The data did not indicate that asparagine biosynthesis in alfalfa nodules has an absolute requirement for glutamine. Radioactivity in the xylem sap, derived from nodule ¹⁴CO₂ fixation, was markedly decreased by treating nodulated roots with aminooxyacetate, methionine sulfoximine, and azaserine. Inhibitors decreased the [¹⁴C]aspartate and [¹⁴]asparagine content of xylem sap by greater than 80% and reduced the total amino nitrogen content of xylem sap (including nonradiolabeled amino acids) by 50 to 80%. Asparagine biosynthesis in alfalfa nodules and transport in xylem sap are dependent upon continued aminotransferase activity and an uninterrupted assimilation of ammonia via the glutamine synthetase/glutamate synthase pathway. Continued assimilation of ammonia apparently appears crucial to continued root nodule CO₂ fixation in alfalfa.     

The incorporation of nitrogen into products of recent photosynthesis in Anabaena cylindrica Lemm.

In vivo tracer studies with ¹⁴C have been performed to help determine pathways of incorporation of newly assimilated nitrogen into N₂-fixing cells of Anabaena cylindrica. After photosynthesis in Ar:O₂:¹⁴CO₂ for 30 min, the addition of N₂ or NH ₄ ⁺ resulted in increased rates of ¹⁴CO₂-incorporation both in the light and dark, and in increased incorporation of ¹⁴C into amino acids at the expense of sucrose and sugar phosphates. Evidence of enhanced sucrose catabolism and increased pyruvate kinase activity was obtained on adding nitrogen, and, of the ¹⁴C-labelling entering the tricarboxylic acid cycle, more appeared in citrate and 2-oxoglutarate than in malate and oxaloacetate. The kinetics of ¹⁴C-incorporation into various amino acids suggest that in the light and dark the most important route of primary ammonia assimilation involves glutamine synthetase and that glutamate, aspartate, glycine and probably alanine are formed secondarily from glutamine.     

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.

     

Metabolic fate of fumarate, a side product of the purine salvage pathway in the intraerythrocytic stages of Plasmodium falciparum

In aerobic respiration, the tricarboxylic acid cycle is pivotal to the complete oxidation of carbohydrates, proteins, and lipids to carbon dioxide and water. Plasmodium falciparum, the causative agent of human malaria, lacks a conventional tricarboxylic acid cycle and depends exclusively on glycolysis for ATP production. However, all of the constituent enzymes of the tricarboxylic acid cycle are annotated in the genome of P. falciparum, which implies that the pathway might have important, yet unidentified biosynthetic functions. Here we show that fumarate, a side product of the purine salvage pathway and a metabolic intermediate of the tricarboxylic acid cycle, is not a metabolic waste but is converted to aspartate through malate and oxaloacetate. P. falciparum-infected erythrocytes and free parasites incorporated [2,3-(14)C]fumarate into the nucleic acid and protein fractions. (13)C NMR of parasites incubated with [2,3-(13)C]fumarate showed the formation of malate, pyruvate, lactate, and aspartate but not citrate or succinate. Further, treatment of free parasites with atovaquone inhibited the conversion of fumarate to aspartate, thereby indicating this pathway as an electron transport chain-dependent process. This study, therefore, provides a biosynthetic function for fumarate hydratase, malate quinone oxidoreductase, and aspartate aminotransferase of P. falciparum.     

The pathway of carbon dioxide assimilation in rhodospirillum rubrum grown in turbidostat continuous-flow culture

Summary A comparison of light and dark short-term incorporation of [¹⁴C]-carbon dioxide by Rhodospirillum rubrum grown in turbidostat continuous-flow culture at two different steady states on medium containing malate has shown that the labelling of phosphate esters was the main light-dependent process. Thus, the reductive pentose phosphate cycle appears to be the major pathway of carbon dioxide assimilation in the light under these growth conditions.The labelling of glutamate was also light-dependent and was most marked in the most rapidly growing steady state culture.The assimilated [¹⁴C]carbon was transferred to metabolites of the tricarboxylic acid cycle, particularly C₄-dicarboxylic acids, and the transfer involved additional carboxylations which were not light-dependent. The activity of these reactions accounted for initial high rates of carbon dioxide assimilation in the dark.In the dark assimilated [¹⁴C]carbon accumulated in succinate.     

Compartmentation of Organic Acids in Corn Roots. III. Utilization of Exogenously Supplied Acids

The rates of utilization of exogenously supplied ¹⁴C labeled acids by corn roots was compared to the utilization of these acids generated endogenously in the mitochondria from acetate-³H. ¹⁴C-labeled citrate, pyruvate, succinate, glutamate or aspartate were supplied with acetate-³H in a 15 minute pulse and the ¹⁴C and ³H contents of extracted acids were measured over a 4 hour period. It was found, in contrast to previous experiments with malate, that these exogenously added acids were used as rapidly as the endogenous forms. Apparently, therefore, these acids penetrate readily into the mitochondria and do not enter cytoplasmic pools which are not in ready equilibrium with those in the mitochondria. Small amounts of labeled glutamate were produced from succinate-2,3-³H by corn root tissue. Since glutamate would not be expected to be labeled by reactions of the tricarboxylic acid cycle it was concluded that it was produced rather directly from succinate. The minor pool of glutamate generated in this way retained its radioactivity while that generated in the cycle was rapidly lost. An extra-mitochondrial location of this pool of glutamate is therefore suggested.     

Einfluß kurzer Dunkelperioden auf CO2-Aufnahme und Phosphoenolpyruvat-Carboxylierung während der Photosynthese-Induktion bei Chlorella vulgaris

Zusammenfassung Nach einer Dunkelperiode von 40 min und 40 sec wurden die CO₂-Aufnahme und die ¹⁴C-markierten Produkte während der Photosynthese-Induktion bei Chlorella vulgaris (211-11f) bestimmt. Die mit Preßluft (0,03 Vol.-% CO₂) begasten Algen sind bei +27°C kultiviert und bei +10° oder +25°C gemessen worden. Ein Induktionseffekt der photosynthetischen CO₂-Aufnahme konnte nur nach einer längeren Dunkelperiode (>3 min) beobachtet werden. Unter diesen Bedingungen wurde ¹⁴CO₂ am Anfang der Belichtung in Malat, Aspartat und 3-Phosphoglycerat eingebaut. Nach einer kurzen Dunkelperiode (40 sec) waren zu Beginn der Belichtung vor allem die Produkte des Calvin-Cyclus markiert. Die Wirkung von Intermediaten auf die Ausbildung der Induktionseffekte wird diskutiert.

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Effect of short dark periods on CO₂ uptake and carboxylation of phosphoenolpyruvate during the photosynthetic induction period in Chlorella vulgaris

Summary CO₂ exchange, ¹⁴CO₂ fixation and ¹⁴C labelled products of Chlorella vulgaris (strain 211-11f) were studied during the photosynthetic induction period at +10° and +25°C after a dark period of 40 min and 40 sec. The algae were grown under normal aerated conditions (0.03 vol.-% CO₂) at +27°C. Transient changes in CO₂ uptake, measured with an infrared gas analyzer, could be observed only after a dark period of >3 min; no such changes occurred after a dark period of 40 sec. The autoradiographic studies of the kinetics of the appearance of labelled products at +10° and +25°C showed that after a long dark period (40 min) at the beginning of illumination ¹⁴CO₂ was incorporated into malate, aspartate and 3-phosphoglycerate. Under these conditions, the intermediates of the Calvin cycle were labelled after 30 sec (+25°C) or 2 min (+10°C) of photosynthesis. After a dark period of 40 sec (at +10° and +25°C), however, ¹⁴C incorporation into malate and aspartate was rather low at the beginning of illumination; moreover, the intermediates of the Calvin cycle appeared earlier and were more strongly labelled after this short dark period. The results are discussed with reference to the influence of intermediates on the formation of the transient changes of CO₂ uptake in Chlorella.

     

Mapping the carbon reduction cycle: a personal retrospective

The photosynthetic carbon reduction cycle was elucidated through the use of ¹⁴CO₂ during photosynthesis to label metabolic intermediates. Mapping and proof of the cycle required identification of labeled metabolites, observation of changes in levels of labeled metabolites during transitions from light to dark and from high to low CO₂ levels, determination of intramolecular distribution of ¹⁴C within the metabolites after a few seconds of photosynthesis with ¹⁴CO₂, and estimation of metabolite concentrations, used to calculate true free energy changes at each step in the cycle. This revised version was published online in August 2006 with corrections to the Cover Date.     

High CO2 partial pressure effects on dark and light CO2 fixation and metabolism in Vicia faba leaves

Stomatal opening on Vicia faba can be induced by high CO₂ partial pressures (10.2%) in dark as well as in light. Stomatal aperture was measured in both cases with a hydrogen porometer. The distribution of ¹⁴C among early products of photosynthesis was studied. Comparisons are made with carboxylations occurring when stomata were open in the dark with CO₂-free air and in light with 0.034% CO₂. Results showed that in high CO₂ partial pressure in light, less radioactivity was incorporated in Calvin cycle intermediates and more in sucrose. carboxylations and photorespiration seemed to be inhibited. In the dark in both CO₂ conditions, ¹⁴C incorporation was found in malate and aspartate but also in serine and glycerate in high CO₂ conditions. In light these changes in metabolic pathways may be related with the deleterious effects recorded on leaves after long-term expositions to high partial pressure of CO₂.Abbreviations DHAP dihydroxyacetone phosphate - PEP phosphonenolpyruvate - PEPCK phosphonenolpyruvatecarboxykinase - PGA 3-phosphoglyceric acid - RUBPc ribulose 1,5-bisphosphate carboxylase