Effect of tolbutamide on the metabolism of Tetrahymena

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

Metabolic Interactions Between Glucose, Glycerol, Alanine and Acetate in Leishmania braziliensis panamensis Promastigotes

¹³C-nuciear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of ¹⁴CO₂ production from ¹⁴C-labeIed substrate. Cells incubated with [2-¹³C]glycerol released acetate, succinate and D-lactate in addition to CO₂. Cells incubated with acetate released only CO₂. More succinate C-2/C-3 than C-l/C-4 was released from both [2-¹³C]glycerol and [2-¹³C]glucose, indicating that succinate was formed predominantly by CO₂ fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of ¹⁴CO₂ production from [l,(3)-¹⁴C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased ¹⁴CO₂ production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of ¹⁴CO₂ production from [U-¹⁴C]- and [l-¹⁴C]alanine by about 20%. This is consistent with rapid equilibration of alanine with pyruvate derived from glucose and yet little decrease in the specific activity of the large alanine pool.     

Interactions between Ethylene, CO(2), and ABA on GA(3)-Induced Amylase Synthesis in Barley Aleurone Tissue

Gibberellic acid-induced synthesis and release of α-amylase in barley aleurone tissue was inhibited by abscisic acid. This inhibition was relieved by simultaneous application of ethylene ranging in concentration from 0.1 to 100 microliters per liter. When CO₂ was applied, it eliminated the effect of 0.1 microliter per liter ethylene and reimposed the abscisic acid inhibition. All concentrations of CO₂ tested from 400 to 10⁵ microliters per liter counteracted the effect of 0.1 microliter per liter ethylene, but had no observable effect on any higher concentration of ethylene. The results indicate that some processes necessary for embryo growth may be subject to regulation by ethylene and carbon dioxide at naturally occurring concentrations of the gases.     

Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii

Chloroplastic respiration was monitored by measuring ¹⁴CO₂ from ¹⁴C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of ¹⁴CO₂ evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K_m for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of ¹⁴CO₂ was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO₂ evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO₂ evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH₄Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO₂ and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.     

Acetate and carbon dioxide assimilation by Desulfovibrio vulgaris (Marburg), growing on hydrogen and sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source and acetate plus CO₂ as the sole carbon sources. The incorporation of U-¹⁴C acetate into alanine, aspartate, glutamate, and ribose was studied. The labelling data show that alanine is synthesized from one acetate (C-2 + C-3) and one CO₂ (C-1), aspartate from one acetate (C-2 + C-3) and two CO₂ (C-1 + C-4), glutamate from two acetate (C-1–C-4) and one CO₂ (C-5), and ribose from 1.8 acetate and 1.4 CO₂. These findings indicate that in Desulfovibrio vulgaris (Marburg) pyruvate is formed via reductive carboxylation of acetyl-CoA, oxaloacetate via carboxylation of pyruvate or phosphoenol pyruvate, and -ketoglutarate from oxaloacetate plus acetyl-CoA via citrate and isocitrate. Since C-5 of glutamate is derived from CO₂, citrate must have been formed via a (R)-citrate synthase rather than a(S)-citrate synthase. The synthesis of ribose from 1.8 mol of acetate and 1.4 mol of CO₂ excludes the operation of the Calvin cycle in this chemolithotrophically growing bacterium.     

Effect of a Brief CO(2) Exposure on Ethylene Production

Ethylene production and respiration by Granny Smith apples were inhibited by treatment with 20% CO₂ for 2 hours. A similar effect was observed in tissue slices when treated at either 0 or 25°C.

The inhibition continued even after an extended aeration period. There is also an inhibition of ethylene emission in tissue slices incubated with exogenous 1-aminocyclopropane-1-carboxylic acid (ACC).

In general, CO₂ treatment increased the ACC content of the tissue. These observations are consistent with the idea the action of CO₂ is directed toward the enzyme system responsible for the conversion of ACC into ethylene.

     

Effect of 4-Pentenoic Acid on Intermediate Metabolism of Tetrahymena*

SYNOPSIS. The growth of Tetrahymena pyriformis strain HSM was strongly inhibited by 4-pentenoic acid. Supplementing the medium with acetate reversed the growth inhibition, but pyruvate was ineffective. Glycogen content was much lower in cells grown with 4-pentenoic acid than in controls; this effect was not reversed by acetate or by pyruvate. There was little effect of 4-pentenoic acid on the incorporation of label from [1-¹⁴C]acetate, [2-¹⁴C]glycerol, [1-³⁴]ribose, [U-¹⁴C]fructose, or [1-¹⁴C]glucose into CO₂, but incorporation of label into glycogen was inhibited, the strongest inhibition being on acetate and the weakest (～ 20%) on ribose, fructose, and glucose. A 3-compartment model for quantitation of labeled acetyl CoA fluxes was shown to be applicable to Tetrahymena grown in the presence of 4-pentenoic acid, and experiments were performed to establish the flux of [1-¹⁴C]acetyl CoA into glycogen, lipids, CO₂, glutamate, and alanine. It was evident from the results of these experiments that 4-pentenoic acid did not appreciably inhibit β-oxidation or lipogenesis, but markedly decreased the glyconeogenic flux of labeled acetyl-CoA from the peroxismal and outer mitochondrial compartments. At least 2 mechanisms have been proposed for the action of 4-pentenoic acid: (a) reduction of the levels of acetyl CoA or free CoA and (b) direct inhibition of enzymes by 4-pentenoyl CoA or its metabolites. Although 4-pentenoic acid has little effect on acetyl-CoA metabolism in the inner mitochondrial compartment, the present data suggest that the flux through the outer mitochondrial compartment of acetyl-CoA derived from pyruvate is inhibited largely by the first, and that the glyconeogenic flux of acetyl-CoA is inhibited largely by the 2nd mechanism.     

Ethylene as a regulator of senescence in tobacco leaf discs

The regulatory role of ethylene in leaf senescence was studied with excised tobacco leaf discs which were allowed to senesce in darkness. Exogenous ethylene, applied during the first 24 hours of senescence, enhanced chlorophyll loss without accelerating the climacteric-like pattern of rise in both ethylene and CO₂, which occurred in the advanced stage of leaf senescence. Rates of both ethylene and CO₂ evolution increased in the ethylene-treated leaf discs, especially during the first 3 days of senescence. The rhizobitoxine analog, aminoethoxy vinyl glycine, markedly inhibited ethylene production and reduced respiration and chlorophyll loss. Pretreatment of leaf discs with Ag⁺ or enrichment of the atmosphere with 5 to 10% CO₂ reduced chlorophyll loss, reduced rate of respiration, and delayed the climacteric-like rise in both ethylene and respiration. Ag⁺ was much more effective than CO₂ in retarding leaf senescence. Despite their senescence-retarding effect, Ag⁺ and CO₂, which are known to block ethylene action, stimulated ethylene production by the leaf discs during the first 3 days of the senescing period; Ag⁺ was more effective than CO₂. The results suggest that although ethylene production decreases prior to the climacteric-like rise during the later stages of senescence, endogenous ethylene plays a considerable role throughout the senescence process, presumably by interacting with other hormones participating in leaf senescence.     

Role of Ethylene Metabolism in Amaranthus retroflexus

¹⁴C-Ethylene was metabolized by etiolated pigweed seedlings (Amaranthus retroflexus L.) in the manner similar to that observed in other plants. The hormone was oxidized to ¹⁴CO₂ and incorporated into ¹⁴C-tissue components. Selected cyclic olefins with differing abilities to block ethylene action were used to determine if ethylene metabolism in pigweed is necessary for ethylene action. 2,5-Norbornadiene and 1,3-cyclohexadiene were effective inhibitors of ethylene action at 800 and 6400 microliters per liter, respectively, in the gas phase, while 1,4-cyclohexadiene and cyclohexene were not. However, all four cyclic olefins inhibited the incorporation and conversion of ¹⁴C-ethylene to ¹⁴CO₂ by 95% with I₅₀ values below 100 microliters per liter. The results indicate that total ethylene metabolism does not directly correlate with changes in ethylene action. Additionally, the fact that inhibition of ethylene metabolism by the cyclic olefins did not result in a corresponding increase in ethylene evolution indicates that ethylene metabolism does not serve to significantly reduce endogenous ethylene levels.     

Carbohydrates Stimulate Ethylene Production in Tobacco Leaf Discs : II. Sites of Stimulation in the Ethylene Biosynthesis Pathway

Galactose, sucrose, and glucose (50 millimolar) applied to tobacco leaf discs (Nicotiana tabacum L. cv `Xanthi') during a prolonged incubation (5-6 d) markedly stimulated ethylene production which, in turn, could be inhibited by aminoethoxyvinylglycine (2-amino-4-(2′-aminoethoxy)-trans-3-butenoic acid) (AVG) or Co²⁺ ions. These three tested sugars also stimulated the conversion of l-[3,4-¹⁴C]methionine to [¹⁴C]1-amino-cyclopropane-1-carboxylic acid (ACC) and to [¹⁴C]ethylene, thus indicating that the carbohydrates-stimulated ethylene production proceeds from methionine via the ACC pathway. Sucrose concentrations above 25 mm considerably enhanced ACC-dependent ethylene production, and this enhancement was related to the increased respiratory carbon dioxide. However, sucrose by itself could directly promote the step of ACC conversion to ethylene, since low sucrose concentrations (1-25 mm) enhanced ACC-dependent ethylene production also in the presence of 15% CO₂.     

Purine and pyrimidine biosynthesis in methanogenic bacteria

Following long-term labeling with [1-¹³C]acetate, [2-¹³C]acetate, ¹³CO₂, H¹³COOH, or ¹³CH₃OH, NMR spectroscopy was used to determine the labeling patterns of the purified ribonucleosides of Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii. Major differences were observed among the methanogens studied, specifically at carbon positions 2 and 8 of the purines, positions at which one-carbon carriers are involved during synthesis. In Methanospirillum hungatei and Methanosarcina barkeri, the labcl at both positions came from carbon atom C-2 of acetate, as predicted from known eubacterial pathways, whereas in Methanococcus voltae and Methanobacterium bryantii both originated from CO₂. In Methanosphaera stadtmanae grown in the presence of formate, the C-2 of purines originated exclusively from formate and the C-8 was labeled by the C-2 of acetate. When grown in media devoid of formate, the C-2 of the purine ring originated mainly from the C-2 of acetate and in part from CH₃OH. In Methanobrevibacter smithii grown in the presence of formate, C-2 and C-8 of purines were derived from CO₂ and/or formate. The labeling patterns obtained for pyrimidines are consistent with the biosynthetic pathways common to eubacteria and eucaryotes.Abbreviations CODH Carbon monoxide dehydrogenase - FH₄ tetrahydrofolate - H₄MPT tetrahydromethanopterin Issued as NRCC Publication No. 37383     

β-Poly(l-malate) production by Physarum polycephalum

β-Poly(l-malate) (PMLA) production in Physarum polycephalum has been followed by using d-[1-¹³C]glucose and Ca¹³CO₃. Nuclear magnetic resonance studies of PMLA showed that the ¹³C label from [1-¹³C]glucose was incorporated in the presence of CaCO₃ into positions C-3 (-CH₂-) and C-4 (-CO-) of the l-malate repeating unit of PMLA. The ¹³C label from Ca¹³CO₃ was incorporated into position C-4 and indicated that not only the endogenous CO₂ but also the exogenous CO₂ from CaCO₃ served significantly as a carbon source for PMLA production. In the absence of CaCO₃, the ¹³C labeling pattern of PMLA from d-[1-¹³C]glucose was almost indistinguishable from that for the natural abundance ¹³C-NMR spectrum of the polymer. These results indicated that l-malate used for PMLA production is synthesized either via carboxylation of pyruvate and reduction of oxaloacetate in the presence of CaCO₃ or via the oxidative tricarboxylic acid (TCA) cycle in the absence of CaCO₃. Avidin strongly inhibited the formation of l-malate via carboxylation; the ¹³C labeling pattern of PMLA in the presence of CaCO₃ was almost identical with that for the natural abundance spectrum when avidin was added, indicating that l-malate utilized for PMLA production was supplied under this condition by the oxidative TCA cycle. Received: 16 March 1999 / Received revision: 5 May 1999 / Accepted: 7 May 1999     

Ethylene formation in Pisum sativum and Vicia faba protoplasts

Protoplasts isolated from leaves of peas (Pisum sativum L.) and of Vicia faba L. produced 1-aminocyclopropane-1-carboxylic acid (ACC) from endogenous substrate. Synthesis of ACC and conversion of ACC to ethylene was promoted by light and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and carbonyl cyanide m-chlorophenylhydrazone. Aminoethoxyvinylglycine inhibited ethylene synthesis to a minor extent when given during incubation of the protoplasts but was very effective when added both to the medium in which the protoplasts were isolated and to the incubation medium as well. Radioactivity from [U-¹⁴C]methionine was incorporated into ACC and ethylene. However, the specific radioactivity of the C-2 and C-3 atoms of ACC, from which ethylene is formed, increased much faster than the specific radioactivity of ethylene. It appears that ACC and ethylene are synthesized in different compartments of the cell and that protoplasts constitute a suitable system to study this compartmentation.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Acetate synthesis from 2 CO2 in acetogenic bacteria: is carbon monoxide an intermediate?

Cultures of Acetobacterium woodii and Clostridium thermoaceticum growing on fructose or glucose, respectively, were found to produce small, but significant amounts of carbon monoxide. In the gas phase of the cultures up to 53 ppm CO were determined. The carbon monoxide production was completely inhibited by 1 mM cyanide. Cultures and cell suspensions of both acetogens incorporated ¹⁴CO specifically into the carboxyl group of acetate. This CO fixation into C₁ of acetate was unaffected by cyanide (1 mM). The findings are taken to indicate that CO (in a bound form) is the physiological precursor of the C₁ of acetate in acetate synthesis from CO₂. The cyanide inhibition experiments support the hypothesis that the cyanide-sensitive carbon monoxide dehydrogenase may serve to reduce CO₂ to CO rather than to incorporate the carbonyl into C₁ of acetate.     

The role of carbon dioxide in glucose metabolism of Bacteroides fragilis

The effect of CO₂ concentration on growth and glucose fermentation of Bacteroides fragilis was studied in a defined mineral medium. Batch culture experiments were done in closed tubes containing CO₂ concentrations ranging from 10% to 100% (with appropriate amounts of bicarbonate added to maintain the pH at 6.7). These experiments revealed that CO₂ had no influence on growth rate or cell yield when the CO₂ concentration was above 30% CO₂ (minimum available CO₂–HCO ₃ ^- , 25.5 mM), whereas a slight decrease in these parameters was observed at 20% and 10% CO₂ (available CO₂–HCO ₃ ^- , 17 and 8.5 mM, respectively). If CO₂–HCO ₃ ^- concentrations were below 10 mM, the lag phase lengthened and a decrease in maximal growth rate and cell yield were observed. The amount of acetate made decreased, while d-lactate concentration increased. A net production of CO₂ allowed growth under conditions of extremely low concentrations of added CO₂.When B. fragilis was grown in continuous culture with 100% CO₂ or 100% N₂, the dilution rate influenced the concentrations of acetate, succinate, propionate, d-lactate, l-malate and formate formed. Decreasing the dilution rate favored propionate and acetate production under both conditions. When the organism was grown with 100% N₂, the amount of propionate formed was greater than the amount of succinate formed at all dilution rates. Except at slow dilution rates the reverse was true when 100% CO₂ was used. B. fragilis was unable to grow at dilution rates faster than 0.154 h^-1 when grown with 100% N₂; the Y _glc ^max was 67.9 g DW cells/mol glucose and m _s was 0.064 mmol glucose/g DW·h. If the gas atmosphere was 100% CO₂ the organism was washed out of the culture when the dilution rate exceeded 0.38 h^-1; the Y _glc ^max was 59.4 g DW cells/mol glucose and m _s was 0.094 mmol glucose/g DW·h.Measurement of the phosphoenolpyruvate (PEP) carboxykinase (E.C. 4.1.1.49) with whole, permeabilized cells of B. fragilis showed an increase of specific enzyme activity with decreasing CO₂ concentrations. The mechanisms used by B. fragilis to adjust to low levels of CO₂ are discussed.     

Ethylene and in vitro rooting of rose shoots

Effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene biosynthesis inhibitor, (CoCl₂), and inhibitor of ethylene binding to receptors, 1-methylcyclopropene (1-MCP), on ethylene production and rooting in shoot culture of Rosa hybrida L. cv. Alba meidiland were studied. Additionally, effect of ethylene removal by KMnO₄ and HgClO₄ on rooting was tested. ACC increased ethylene production and delayed root formation, decreased the number of roots per shoot and inhibited root growth. In contrast, inhibition of ethylene production by CoCl₂ accelerated root emergence, and increased the number of roots per shoot. Likewise, removing ethylene from the ambient atmosphere improved root emergence and, increased root number of per shoot and markedly inhibited root growth. Blocking the ethylene receptors by 1-MCP increased ethylene level in the ambient atmosphere and increased both emergence and root formation. Both ethylene biosynthesis and action are involved in the control of rooting. Ethylene concentration in glass jars was too high for root emergence and formation, but was appropriate for root growth. CoCl₂ or 1-MPC can be recommended for regulation of rooting in rose shoot culture, since both emergence and number of roots were improved but root growth was not inhibited.     

Ethylene and wound-induced cyanide-resistant respiration climaxes in potato: The synergistic role of CO2 and the selective role of lycorine

When treated with ethylene in O₂, conditioned potato (Solanum tuberosum L. cv. Russet Burbank) tubers – that is, tubers kept at room temperature for 10 days or more – yield slices that are CN^? resistant. Ten % CO₂ in the gas mixture not only synergizes the effect of ethylene, but replaces the need for conditioning as well. The response to CO₂ is more pronounced with increasing time from harvest. By contrast fresh slices from untreated tubers are CN^? sensitive, as are slices from tubers incubated in O₂ or O₂ plus CO₂. The suggestion is made that CN^? resistance is constitutive, and that treatment with ethylene/CO₂ in O₂ confers on potato tuber tissue a resistance to the extensive degradation of membrane phospholipids that normally attends slicing and leads to the loss of CN^? resistance. In this connection respiration inhibition by imidazole, an inhibitor of fatty acid α-oxidation, is extensive in slices of untreated tubers, and sharply diminished in slices of ethylene-treated tubers in proportion to their CN^? resistance. The coextensive rise of respiration rate and CN^? resistance in aged potato slices has led to the presumption that the CN^?-resistant path mediates the respiration climax. Accordingly the alkaloid, lycorine, has been considered to inhibit the development of CN^? resistance in aging potato slices because it curtails the wound-induced respiration. A comparison was carried out on the effect of lycorine on CN^?-sensitive and CN^?-resistant fresh slices – the latter obtained from ethylene/CO₂-treated tubers. Lycorine suppressed the development of the wound-induced respiration without restricting the development of CN^? resistance.     

Autotrophic synthesis of activated acetic acid from two CO2 in Methanobacterium thermoautotrophicum

In a previous study with Methanobacterium thermoautotrophicum evidence was presented that methanogenesis and autotrophic synthesis of activated acetic acid from CO₂ are linked processes. In this study one-carbon metabolism was investigated with growing cultures and in vitro.Serine was shown to be converted into glycine and activated formaldehyde, but only traces of label from [¹⁴C-3] of serine appeared in biosynthetic one-carbon positions. This seeming discrepancy could be explained if the same activated formaldehyde is an intermediate in biosynthesis and in methanogenesis from CO₂. This hypothesis was supported by demonstrating that [¹⁴C-3] of serine and [¹⁴C] formaldehyde were rapidly converted into methane, but a small portion of the label was also specifically incorporated into the methyl group of acetate. Methane and acetate synthesis in vitro were similarly stimulated by various compounds. These experiments indicate that the methyl of acetate and methane share common one-carbon precursor(s), i.e. methylene tetrahydromethanopterin, which can also be formed enzymatically from C-3 of serine or chemically from formaldehyde.Propyl iodide 20–40 M) and methyl iodide (1–3 M) completely inhibited growth in the dark. This effect was abolished by light. Methane formation was hardly affected. When ¹⁴CH₃I was applied at an only slightly inhibitory concentration, ¹⁴C was incorporated into the methyl of acetate. In vitro, similar effects on [¹⁴C] acetate formation from ¹⁴CO₂ or from [¹⁴C-3] of serine were observed, except that methyl iodide did not inhibit, but even stimulated acetate synthesis. These experiments indicate that a corrinoid is involved in acetate synthesis and probably not in methanogenesis from CO₂; the metal is light-reversibly alkylated and functions in methyl transfer to the acetate methyl.     

Total synthesis of acetyl coenzyme a involved in autotrophic CO2 fixation inAcetobacterium woodii

The Gram positive anaerobeAcetobacterium woodii is able to grow autotrophically with a mixture of H₂ and CO₂ as the energy and carbon source. The question, by which pathway CO₂ is assimilated, was studied using long term isotope labeling.Autotrophically growing cultures produced acetate parallel to cell proliferation, and, when U-[¹⁴C]acetate was present as tracer, incorporated radioactivity into all cell fractions. The specific radioactivity and the label positions were determined for those representative cell compounds which biosynthetically originated directly from acetyl CoA (N-acetyl groups), pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), and hexosephosphates (glucosamine). Per mol compound the same amount of labeled acetate was incorporated into N-acetyl groups, alanine (C-2, C-3), aspartate (C-2, C-3), and twice the amount into glutamate (C-2, C-3, C-4, C-5) and into glucosamine. Consequently, the unlabeled carbon atoms of the C₃–C₆ compounds must have been derived from CO₂ by carboxylation subsequent to acetyl CoA synthesis. When 0.2 mM 2-[¹⁴C]pyruvate was added to autotrophically growing cultures, also a substantial amount of radioactivity was incorporated. Two important differences in comparison to the acetate experiment were observed: The N-acetyl groups were almost unlabeled and glutamate contained the same specific radioactivity as alanine or aspartate.These data showed that acetyl CoA is the central intermediate for biosynthesis and excluded the operation of the Calvin cycle inA. woodii. The results were consistent with the operation of a different autotrophic CO₂ fixation pathway in which CO₂ is converted into acetyl CoA by total synthesis via methyltetrahydrofolate; acetyl CoA is then further reductively carboxylated to pyruvate.     

Carbon Dioxide and Ethylene Control of Spore Germination in Onoclea sensibilis L

Regulation of spore germination in the fern Onoclea sensibilis L. was investigated by applying CO₂ alone and in combination with ethylene. Sterile spores were sown aseptically on Knops solution in loosely capped culture tubes, enclosed individually in 2-liter chambers, and grown under continuous white light. When maintained in enclosed containers with the ethylene-absorbent mercuric perchlorate and with atmospheres enriched up to 2% CO₂ (v/v), spores germinated without any inhibition. Higher levels of applied CO₂ were progressively inhibitory. Inhibition by CO₂ was reversible. When CO₂ was permitted to escape and spores were exposed subsequently to ambient laboratory air, recovery from inhibition occurred within 48 hours. Also, inhibition by CO₂ was specific, since the same degree of inhibition resulted regardless of whether spores were treated with exogenous CO₂ for 48, 72, or 96 hours. The effect on germination of 1 μl/l added ethylene depended upon the amount of applied CO₂. When containers of KOH were enclosed and ambient CO₂ was absorbed, inhibition of germination by 1 μl/l exogenous ethylene was 90%. When CO₂ was applied in concentrations from 0.25 to 1.0% (v/v), CO₂ increasingly antagonized the inhibitory action of 1 μl/l added ethylene. Thus, photoinduced germination of spores was regulated by competitively interacting levels of CO₂ and ethylene.