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.     

The loss of carbon-20 in C19-gibberellin biosynthesis in a cell-free system from Pisum sativum L.

The fate of the carbon-20 atom in gibberellin (GA) biosynthesis was studied in a cell-free system from Pisum sativum. This carbon atom is lost at the aldehyde stage of oxidation when C₂₀-GAs are converted to C₁₉-GAs. Gibberellin A₁₂ labeled with ¹⁴C at C-20 was prepared from [3-¹⁴C]mevalonic acid with a cell-free system from Cucurbita maxima and incubated with the pea system. Analysis of the gas and aqueous phases showed that ¹⁴CO₂ was formed at the same rate and in nearly equivalent amounts as ¹⁴C-labeled C₁₉-GAs whereas [¹⁴C]formic acid and [¹⁴C]formaldehyde were not detectable. The possibility that C-20 had been lost as formic acid which had then been converted to CO₂ was investigated by control incubations with [¹⁴C]formic acid. The rate of release of ¹⁴CO₂ from [¹⁴C]formic acid was only one fiftieth of the rate of ¹⁴CO₂ release from [¹⁴C]GA₁₂ as the substrate. We conclude that in the formation of C₁₉-GAs from C₂₀-GAs, the C-20 is removed directly as CO₂.Abbreviations GA_n Gibberellin A_n     

Microbial degradation of ethylenediaminetetraacetate in soils and sediments

[¹⁴C]ethylenediaminetetraacetate (EDTA) was shown to be slowly biodegraded to ¹⁴CO₂ in soils and sediments under aerobic conditions and by microorganisms in mixed liquid culture. EDTA chelates of Cu, Cd, Zn, Mn, Ca, and Fe added to soil were equally degraded, while Ni-EDTA was degraded more slowly.     

Vitamin K dependent formation of gamma-carboxyglutamate residues in tumor microsomes

Vitamin K stimulated the incorporation of ¹⁴C into proteins when microsomes from melanoma, mammary gland, mast cell and lymphoma tumors were incubated with Na₂¹⁴CO₃. The ¹⁴C label in the [¹⁴C] proteins was identified as [¹⁴C] γ-carboxyglutamate (Gla), which is formed by carboxylation of glutamic acid residues. Carboxylation in tumor microsomes ranged from 2 to 19% of the carboxylation in normal liver microsomes per mg of microsomal protein. Carboxylation in microsomes was completely blocked by 10 μM Warfarin. SDS-polyacrylamide gel analysis of the melanoma [¹⁴C] Gla protein(s) revealed one major peak of ¹⁴C with an apparent MW of less than 6,000.     

Oxidation of C1-compounds in Pseudomonas C

Extracts of Pseudomonas C grown on methanol as sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD⁺) and formate dehydrogenase (NAD⁺) were also detected in the extracts.The addition of d-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD⁺ or NADP⁺. In addition, the oxidation of [¹⁴C]formaldehyde to CO₂, by extracts of Pseudomonas C, increased when d-ribulose 5-phosphate was present in the assay mixtures.The amount of radioactivity found in CO₂, was 6.8-times higher when extracts of methanol-grown Pseudomona C were incubated for a short period of time with [1-¹⁴C]glucose 6-phosphate than with [U-¹⁴C]glucose 6-phosphate.These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO₂ both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.     

Validation of the design of feeding experiments involving [(14)C]substrates used to monitor metabolic flux in higher plants

The aim of this study was to examine whether flux through the pathways of carbohydrate oxidation is accurately reflected in the pattern of ¹⁴CO₂ release from positionally labelled [¹⁴C]substrates in conventional radiolabel feeding studies. Heterotrophic cell suspension cultures of Arabidopsis thaliana were used for this work. The presence of an alkaline trap to capture metabolically generated ¹⁴CO₂ had no significant effect on the ratio of ¹⁴CO₂ release from specifically labelled [¹⁴C]substrates, or on the metabolism of [U-¹⁴C]glucose by the cells. Although the amount of ¹⁴CO₂ captured in a conventional time-course study was only about half of that released from a sample acidified at an equivalent time point, the ratios of ¹⁴CO₂ released from different positionally labelled [¹⁴C]glucose and [1-¹⁴C]gluconate were the same in untreated and acidified samples. Less than 5% of radioactivity supplied to the growth medium as [¹⁴C]bicarbonate was incorporated into acid-stable compounds, and there was no evidence for appreciable reassimilation of ¹⁴CO₂ generated intracellularly during oxidation of [1-¹⁴C]gluconate by the cells. It is concluded that the ratio of label captured from specifically labelled [¹⁴C]glucose is a valid and convenient measure of the relative rates of oxidation of the different positional carbon atoms within the supplied respiratory substrate. However, it is argued that failure to compensate for the incomplete absorption of ¹⁴CO₂ by an alkaline trap may distort estimates of respiration that rely on an absolute measure of the amount of ¹⁴CO₂ generated by metabolism.     

Inhibition of glucose utilization for acetylcholine synthesis by cerebrospinal fluid.

Abstract— Replacement of bicarbonate-Locke incubation medium with feline CSF reduced [¹⁴C]ACh formation from [U-¹⁴C]glucose by rat brain mince approx 30%. CSF was obtained from a cannula leading to the cisterna magna of freely moving cats. The component of CSF responsible for inhibition was characterized as a dialyzable heat-stable organic anion. Choline acetyltransferase activity was not altered by CSF. [¹⁴C]ACh synthesis and ¹⁴CO₂ production from [U-¹⁴C]glucose but not from [2-¹⁴C]-pyruvate were inhibited by CSF, suggesting inhibition in the metabolism of glucose to pyruvate. The anionic fraction of human CSF was as potent as that from feline CSF in inhibiting ¹⁴CO₂ production from [U-¹⁴C]glucose. Brain hexokinase was inhibited by the anionic fraction of feline CSF. The inhibition was non-competitive with respect to glucose and uncompetitive with respect to ATP. It is suggested that inhibition of hexokinase by CSF was responsible at least in part for the inhibition of glucose metabolism which resulted in decreased [¹⁴C]ACh synthesis and ¹⁴CO₂ production.     

Mitochondrial compartmentation of metabolic CO2 resulting from its site of origin in relation to urea synthesis

In isolated hepatocytes the entry into urea of metabolic ¹⁴CO₂; derived from [¹⁴C] formate is modified by the addition of dichloroacetate and hydroxypyruvate. An explanation is that this results from changes in the cytoplasmic/mitochondrial pH gradient. ¹⁴CO₂, derived from [1-¹⁴C]alanine enters into urea more readily than ¹⁴CO₂ arising from [1-¹⁴C]glutamate. It is proposed that the difference, which is more than 4-fold, is indicative of a preferred pathway for metabolic CO₂ in liver mitochondria from pyruvate dehydrogenase to carbamoylphosphate synthetase than form oxoglutarate dehydrogenase. Acetazolamide inhibition of carbonic anhydrase is without effect on this observed incorporation into urea.     

Uptake and metabolism of glutamate and aspartate by astroglial and neuronal preparations of rat cerebellum

Astrocytes, neuronal perikarya and synaptosomes were prepared from rat cerebellum. Kinetics of high and low affinity uptake systems of glutamate and aspartate, nominal rates of¹⁴CO₂ production from [U–¹⁴C]glutamate, [U–¹⁴C]aspartate and [1–¹⁴C]glutamate and activities of enzymes of glutamate metabolism were studied in these preparations. The rate of uptake and the nomial rate of production of¹⁴CO₂ from these amino acids was higher in the astroglia than neuronal perikarya and synaptosomes. Activities of glutamine synthetase and glutamate dehydrogenase were higher in astrocytes than in neuronal perikarya and synaptosomes. Activities of glutaminase and glutamic acid decarboxylase were observed to be highest in neuronal perikarya and synaptosomes respectively. These results are in agreement with the postulates of theory of metabolic compartmentation of glutamate while others (presence of glutaminase in astrocytes and glutamine synthetase in synaptosomes) are not. Results of this study also indicated that (i) at high extracellular concentrations, glutamate/aspartate uptake may be predominantly into astrocytes while at low extracellular concentrations, it would be into neurons (ii) production of -ketoglutarate from glutamate is chiefly by way of transamination but not by oxidative deamination in these three preparations and (iii) there are topographical differences glutamate metabolism within the neurons.     

GLUCOSE AND AMINO ACID METABOLISM IN OCTOPUS OPTIC AND VERTICAL LOBES IN VITRO

(1) The metabolism of glucose and amino acids in vitro was compared in the rat cerebral cortex and the optic and vertical lobes of the octopus brain. (2) Specific activities and pool sizes of the five amino acids, glutamate, aspartate, glutamine, alanine and γ-aminobutyric acid (GABA), were determined in octopus and rat brain slices after 2 hr incubation with 10 mm -[U-¹⁴C]glucose, 10 mm -L-[U-¹⁴C]glutamate, and 10mm -^L-[U-¹⁴C]glutamate with added 10 mM-glucose. Amino acid pool sizes were similar in rat and octopus brain, with the exception of alanine, which was higher in the octopus. Generally specific activities were from four- to 20-fold higher in rat brain. With [U-¹⁴C]glucose as substrate, specific activities of GABA and glutamate were highest in rat; those of alanine and glutamine highest in octopus brain. With ^L-[U-¹⁴C]glutamate the specific activities of GABA and aspartate were highest in rat, that of aspartate highest and GABA lowest in octopus. The addition of glucose to ^L-[U-¹⁴C]glutamate as substrate had little effect on the specific activities of any of the amino acids. (3) The uptake of some amino acids was determined by incubation with [U-¹⁴C]amino acids for 2 hr, and ¹⁴CO₂ formation was also measured. The amount of label taken up by octopus was uniformly 20-25 per cent of that found for rat brain. The amount of ¹⁴CO₂, however, differed according to the amino acid. Four times as much ¹⁴CO₂ was generated from alanine by octopus optic lobe and twice as much by the vertical lobe than rat cortex, but from glutamate, only 24 per cent in the optic and 15 per cent in the vertical lobe. No ¹⁴CO₂ was generated from [U-¹⁴C]GABA in the octopus, by contrast with the rat. (4) Activity of some of the enzymes involved in amino acid metabolism was determined in homogenates of rat cortex and octopus optic and vertical lobes, with and without activation by Triton X-100. Enzymic activities in the octopus, with the exception of alanine aminotransferase, were lower than in the rat, and glutamate decarboxylase could not be detected in octopus brain, in the absence of detergent.     

Comparison of oxidative metabolism in starved, fat-fed and carbohydrate-fed rats

1. Rats were starved for 48hr. or fed for 1 week on a high-fat or a high-carbohydrate diet. The effects of these dietary alterations on the rate of production of ¹⁴CO₂ from trace amounts of [U-¹⁴C]glucose, [1-¹⁴C]palmitate or [1-¹⁴C]acetate administered intravenously were studied. 2. The oxidation of [¹⁴C]glucose was most rapid in the carbohydrate-fed condition and was decreased significantly and to the same extent after starvation and after feeding with fat. 3. Under all dietary regimes studied the maximum rate of elimination of ¹⁴CO₂ from [¹⁴C]palmitate occurred within a few minutes after injection, but considerably more was oxidized after starvation and feeding with fat than after feeding with carbohydrate. 4. Alterations in diet had no effect on the oxidation and high recovery of administered [¹⁴C]acetate as ¹⁴CO₂. 5. Graphical analysis showed the presence of several exponential components in the ¹⁴CO₂-elimination curves. 6. In all studies a marked similarity in oxidative pattern was noted between the starved and the fat-fed rat.     

Oxalate metabolism by tobacco leaf discs

The turnover rate of oxalate in leaf discs of Nicotiana tabacum, var Havana Seed, during photosynthesis was estimated to be 1 to 2 micromoles per gram fresh weight per hour. Radioactivity from the enzymic oxidation of [¹⁴C]oxalate rapidly appeared in neutral sugars (mainly sucrose), organic acids (mainly malate), and amino acids. Only 5% of the radioactivity was released to the atmosphere as ¹⁴CO₂, and no formate or formaldehyde could be detected. The metabolism of oxalate was not increased by raising the O₂ concentration from 1% to 21% to 60%, nor was the formation of [¹⁴C]oxalate from [2-¹⁴C]glyoxylate changed under the same conditions as was previously observed in vitro (Havir 1983 Plant Physiol 71: 874-878). While oxalate is not an inert end product of the glycolate pathway, it contributes little to the formation of photorespiratory CO₂.     

GLUCOSE AND AMINO ACID METABOLISM IN SOME INVERTEBRATE NERVOUS SYSTEMS

(1) The in vitro metabolism of [U-¹⁴C]glucose and [U-¹⁴C]glutamate was compared in snail, octopus and locust ganglia, and in rat cerebral cortex. (2) The metabolic patterns are quantitatively similar. The major labelled metabolites formed from glucose or glutamate by rat cortex and the invertebrate systems were CO₂, aspartate, glutamate, glutamine and alanine. γ-Aminobutyric acid (GABA) was formed in substantial amounts only by locust and rat. (3) A much larger proportion of labelled glucose and glutamate was converted to alanine by the invertebrates compared with rat cortex, although ¹⁴CO₂ production was lower. (4) The effect of glucose in reducing aspartate formation and stimulating glutamine formation from [U-¹⁴C]glutamate in mammalian cortex was observed in the locust but not in the molluscs. (5) Labelled citric acid cycle intermediates were formed in substantial quantities from glucose and glutamate only by snail and locust.     

Oxalate synthesis from [14C1]glycollate and [14C1]glycoxylate in the hepatectomized rat

Hepatectomy significantly altered the metabolism of [1-¹⁴C]glyoxylate and [1-¹⁴C]glycollate in the rat. The production of ¹⁴CO₂ was reduced by 47% and 77%–86%, respectively, indicating the involvement of the liver in the oxidation of both substrates. Unidentified intermediates, assumed to be primary glycine, serine and ethanolamine, were also reduced by over 50%, was would be expected from the removal of the aminotransferase enzymes through the hepatectomy. The biosynthesis of [¹⁴C]oxalate from [1-¹⁴C]glycollate was reduced by more than 80% in the hepatectomized rat. This suggests that this oxidation is primarily catalyzed by the liver enzymes, glycolic acid oxidase and glycolic acid dehydrogenase, in the intact rat. The limited formation of [¹⁴C]oxalate from [¹⁴₁]glycollate observed in the hepatectomized rat is probably catalyzed by lactate dehydrogenase or extrahepatic glycolic acid oxidase. Hepatectomy did not significantly alter the rate of formation of [¹⁴C]oxalate from [¹⁴₁]glyoxylate. However, since saturating concentrations of glyoxylate could not be used because of the toxicity of this substrate, the involvement of glycollic acid oxidase in this oxidation reaction in the intact rat can not be ruled out. In the hepatectomized rat, lactate dehydrogenase appears to be the enzyme making the major contribution, although other as yet not identified enzymes may be contributing. The increased deposition of oxalate in the tissues, oxalosis, may result from the shift in oxalate synthesis from the liver to the extrahepatic tissues.     

Glucose metabolism in anaerobic rice seedlings

More than 80% of the radioactivity from [U-¹⁴C]glucose metabolised by anaerobic rice seedlings or by excised roots or coleoptiles was recovered as ethanol plus CO₂; less than 5% was recovered as water-soluble acidic components. Rates of ¹⁴CO₂ formation from [U-¹⁴C]glucose were similar in roots and coleoptiles in both N₂ and air atmospheres. More ¹⁴CO₂ was formed from [U-¹⁴C]glucose than could be accounted for by ethanolic fermentation, and the specific yields of ¹⁴CO₂ from [6-¹⁴C]glucose and [1-¹⁴C]glucose gave unusually high C-6/C-1 ratios (1.7) in the anaerobic coleoptile. The results may indicate that appreciable pentan synthesis occurs in the anaerobic coleoptile.     

Anaerobic formation of succinate from glucose and bicarbonate in resting cells of Leishmania donovani

The cell suspension of Leishmania donovani incorporates ¹⁴CO₂ resulting in the formation of [¹⁴C]-succinic acid under anaerobic conditions. The results showed that the [¹⁴C]-succinate formation from [1-¹⁴C]-glucose is much greater than that from [6-¹⁴C]-glucose. [¹⁴C-pyruvate] takes part in the production of succinic acid under anaerobic conditions without decarboxylation. The anaerobic formation of succinate appears to involve the production of malate, which is then converted to succinate via the reduction of fumarate by the reversal of the tricarboxylic acid cycle. Evidence indicated that the active species in this carboxylation reaction was CO₂ although HCO₃ was active to some extent.     

Glycine,Serine, and Leucine Metabolism in Different Regions of Rat Central Nervous System

We have investigated the glycine, serine and leucine metabolism in slices of various rat brain regions of 14-day-old or adult rats, using [1-¹⁴C]glycine, [2-¹⁴C]glycine, L-[3-¹⁴C]serine and L-[U-¹⁴C]leucine. We showed that the [1-¹⁴C]glycine oxidation to CO₂ in all regions studied occurs almost exclusively through its cleavage system (GCS) in brains of both 14-day-old and adults rats. In 14-day-old rats, the highest oxidation of [1-¹⁴C]glycine was in cerebellum and the lowest in medulla oblongata. In these animals, the L-[U-¹⁴C]leucine oxidation was lower than the [1-¹⁴C]glycine oxidation, except in medulla oblongata where both oxidations were the same. Serine was the amino acid that showed lowest oxidation to CO₂ in all structure studied. In adult rats brains, the highest oxidation of [1-¹⁴C]glycine was in cerebral cortex and the lowest in medulla oblongata. We have not seen difference in the lipid synthesis from both glycine labeled, neither in 14-day-old rats nor in adult ones, indicating that the lipids formed from glycine were not neutral. Lipid synthesis from serine was significantly high than lipid synthesis and from all other amino acids studied in all studied structures. Protein synthesis from L-[U-¹⁴C]leucine was significantly higher than that from glycine in all regions and ages studied.     

Citrate formation by rat lung mitochondrial preparations

Rat lung mitochondrial preparations were incubated in the presence of pyruvate and malate. The principal metabolic products measured were citrate and CO₂. Citrate formation from pyruvate was found to be dependent on the presence of malate. Significant citrate was formed in the presence of isocitrate and the rate of citrate formation was increased by the addition of pyruvate. Small amounts of citrate were formed by lung mitochondrial preparations in the presence of 2-oxoglutarate and succinate only after the addition of pyruvate. The level of acetyl-CoA was significantly greater in the presence of pyruvate than in the presence of pyruvate plus malate. The addition of malate to lung mitochondrial preparations increased ¹⁴CO₂ production from [U-¹⁴C]- and [1-¹⁴C] pyruvate but decreased its production from [2-¹⁴C]- and [3-¹⁴C]-pyruvate. However, malate increased the incorporation of [2-¹⁴C] pyruvate into malate and citrate. A low level of pyruvate-dependent H¹⁴CO₈-incorporation into acid-stable products was observed, principally citrate and malate, but this rate did not exceed 5% of the rate of net citrate formation in the presence of malate and pyruvate. The capacity of rat lung mitochondria to form oxaloacetate from pyruvate alone in vitro is very limited, and would appear to cast doubt on a major role of pyruvate carboxylase in citrate formation. It is concluded that the rate of citrate formation from pyruvate is limited by the availability of intramitochondrial oxaloacetate and the rate of citrate efflux across the mitochondrial membrane.     

Metabolism of glycerol-1,2,3-trimethylsuccinate in rat hepatocytes

The metabolism of glycerol-1,2,3-trimethylsuccinate ester was investigated in rat hepatocytes. The ester displayed a greater nutritional value than D-glucose, as a precursor of either CO₂ or glycogen. In terms of ¹⁴CO₂ production, the value calculated from experiments conducted in the presence of 1.9 mM [U-¹⁴C] glycerol-1,2,3-trimethylsuccinate, glycerol-1,2,3-trimethyl[1,4-¹⁴C] succinate and glycerol- 1,2,3-trimethyl[2,3-¹⁴C] succinate represented about 50 times that found in cells incubated with 1.0 mM D-[U-¹⁴C] glucose. For glycogen synthesis, the results found with the ester were approximately 7-8 times higher than those found with the hexose. A further advantage of the ester over D-glucose consisted in the fact that, at increasing concentrations of these nutrients, a maximal metabolic response may be reached at lower levels of glycerol-1,2,3-trimethylsuccinate than D-glucose. By comparison with previous data obtained in the same experimental model, glycerol-1,2,3-trimethylsuccinate was also found to display a higher nutritional value than the dimethyl ester of succinic acid. It is proposed, therefore, that glycerol-1,2,3-trimethylsuccinate could be used to support ATP generation in cells endangered by an imbalance between the rate of synthesis and hydrolysis of this adenine nucleotide.     

Metabolism of Acetate, Methanol, and Methylated Amines in Intertidal Sediments of Lowes Cove, Maine

The fates and the rates of metabolism of acetate, trimethylamine, methylamine, and methanol were examined to determine the significance of these compounds as in situ methane precursors in surface sediments of an intertidal zone in Maine. Concentrations of these potential methane precursors were generally <3 μM, with the exception of sediments containing fragments of the seaweed Ascophyllum nodosum, in which acetate was 96 μM. [2-¹⁴C]acetate turnover in all samples was rapid (turnover time <2 h), with ¹⁴CO₂ as the primary product. [¹⁴C]trimethylamine and methylamine turnover times were slower (>8 h) and were characterized by formation of both ¹⁴CH₄ and ¹⁴CO₂. Ratios of ¹⁴CH₄/¹⁴CO₂ from [¹⁴C]trimethylamine and methylamine in uninhibited sediments indicated that a significant fraction of these substrates were catabolized via a non-methanogenic process. Data from inhibition experiments involving sodium molybdate and 2-bromoethanesulfonic acid supported this interpretation. [¹⁴C]methanol was oxidized relatively slowly compared with the other substrates and was catabolized mainly to ¹⁴CO₂. Results from experiments with molybdate and 2-bromoethanesulfonic acid suggested that methanol was oxidized primarily through sulfate reduction. In Lowes Cove sediments, trimethylamine accounted for 35.1 to 61.1% of total methane production.