GLUCOSE AND AMINO ACID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELL FRACTIONS IN VITRO

Abstract—

• 1 The metabolism of three substrates, [U-¹⁴C]glucose, [U-¹⁴C]pyruvate and [U-¹⁴C]glutamate has been studied in vitro in neuronal and glial cell fractions obtained from rat cerebral cortex by a density gradient technique.
• 2 The mixed cell suspension, after washing, metabolized glucose and glutamate in a manner essentially similar to the tissue slice. Exceptions were a reduced ability to generate lactate from glucose and alanine from glutamate, and a lowered effect of added glucose in suppressing the production of aspartate from glutamate.
• 3 After 2 hr incubation with [U-¹⁴C]glucose, the concentration of the amino acids glutamate, glutamine, GABA, aspartate and alanine were raised in the neuronal, compared to the glial fraction to 234 per cent, 176 per cent, 202 per cent, 167 per cent and 230 per cent respectively although both were lower than in the tissue slice. Incorporation of radio-activity was absolutely lower in the neuronal fraction, however, and the specific activities of the amino acids were: glutamate 12 per cent, GABA 18 per cent, aspartate 34 per cent, and alanine 33 per cent of those in the glial fraction.
• 4 After the incubation with [U-¹⁴C]pyruvate, the pool size of the amino acids were higher than after incubation with glucose, except for GABA, which was reduced to one-third. The concentrations of the amino acids glutamate, glutamine, GABA, aspartate, and alanine in the neuronal fraction were respectively 46 per cent, 143 per cent, 105 per cent, 97 per cent, and 57 per cent of those in the glial. Thus, with the exception of alanine, the specific activity of the neuronal amino acids compared to the glial was little increased when pyruvate replaced glucose as substrate.
• 5 After 2 hr incubation with [U-¹⁴C]glutamate in the presence of non-radioactive glucose, the pool sizes of all the amino acids were increased in both neuronal and glial fractions, with the exception of neuronal alanine and glial glutamine. The concentrations of the amino acids glutamine, GABA, aspartate and alanine were raised in the neuronal fraction, compared to the glial, to 425 per cent, 187 per cent, 222 per cent, and 133 per cent respectively. The specific activities of all the amino acids were higher than with glucose alone with the exception of alanine, and neuronal GABA. Neuronal glutamine and aspartate had specific activities respectively 102 per cent and 84 per cent of glial.
• 6 An unidentified amino acid, with R_F comparable to that of alanine and specific activity close to that of glutamate, was also present after incubation. It was relatively concentrated in the neuronal fraction.
• 7 The distribution of the enzymes glutamate dehydrogenase, aspartate aminotransferase, glutamate decarboxylase and glutamine synthetase between the cell fractions was studied. With the exception of glutamine synthetase, none of the enzymes was lost from the cell fractions during their preparation. Only 14 per cent of the glutamine synthetase, compared with 75 per cent of total protein, was recovered in the fractions. Of the enzymes, glutamate dehydrogenase activity was 406 per cent, and glutamate synthetase activity 177 per cent in the neuronal fraction compared to the glial in the absence of detergent. In the presence of detergent, glutamate dehydrogenase control was 261 per cent, aspartate aminotransferase activity 237 per cent is the neuronal as compared to the glial fraction.
• 8 Incorporation of radioactivity into acid-insoluble material from either glutamate or pyruvate was twice as high into the neuronal as the glial fraction.
• 9 The extent to which these differences may be extrapolated back to the intact tissue is considered, and certain correction factors calculated. The significance of the observations for an understanding of the compartmentation of amino acid pools and metabolism in the brain, and the possible identification of such compartments, is discussed.

     

METABOLISM OF GLUCOSE, AMINO ACIDS, AND SOME RELATED METABOLITES IN THE BRAIN OF TOADS (BUFO BOREAS) ADAPTED TO FRESH WATER OR HYPEROSMOTIC ENVIRONMENTS

The levels and specific radioactivities (SA) of glucose, lactate, pyruvate, α-oxoglutarate and seven amino acids in the brain of toads adapted to fresh water or to an hyperosmotic environment were analysed at various times (5 min–4 h) after an injection of [U-¹⁴C]glucose into the bloodstream. The concentrations and SA of glucose, lactate and five amino acids in blood plasma also were measured. In addition, the SA of glutamine, glutamate, aspartate and GABA in brain were determined 30 min after an injection of [1,5-¹⁴C]citrate into the cisterna magna. The flow of labelled carbon atoms from glucose to amino acids and related metabolites in the toad brain was qualitatively similar to that in the mammalian brain, but quantitatively less than one-tenth of the rate in the brain of rats. Hyperosmotic adaptation induced a large increase in the levels of glucose and amino acids in the brain without affecting the rate of glucose utilization. The SA of several amino acids relative to the SA of glucose were initially lower in hyperosmotically-adapted toads than in toads adapted to fresh water, presumably because of a greater dilution of isotope by the larger amino acid pools in the hyperosmotically-adapted toads. The rates of synthesis of alanine and glutamine from pyruvate and glutamate, respectively, appeared to increase with hyperosmotic adaptation, but the rate of GABA synthesis from glutamate was unaltered. The SA of α-oxoglutarate and glutamate were similar at all time periods in both groups of toads, an indication that these compounds were interconverted much more rapidly than the rate at which α-oxoglutarate was formed from isocitrate. The SA of lactate in comparison to that of glucose varied but was always considerably lower, even at 4 h after the [¹⁴C]glucose injection. After[U-¹⁴C]glucose, glutamine had a SA lower than that of glutamate, whereas after the injection of [1⁴C]citrate, glutamine was formed with a SA much higher than that of glutamate. Hence, glutamate in the toad brain exhibited metabolic compartmentation similar to that in rat brain.     

Acetyl CoA,a central intermediate of autotrophic CO2 fixation in Methanobacterium thermoautotrophicum

The pathway of autotrophic CO₂ fixation in Methanobacterium thermoautotrophicum has been investigated by long term labelling of the organism with isotopic acetate and pyruvate while exponentially growing on H₂ plus CO₂. Maximally 2% of the cell carbon were derived from exogeneous tracer, 98% were synthesized from CO₂. Since growth was obviously autotrophic the labelled compounds functioned as tracers of the cellular acetyl CoA and pyruvate pool during cell carbon synthesis from CO₂. M. thermoautotrophicum growing in presence of U-¹⁴C acetate incorporated ¹⁴C into cell compounds derived from acetyl CoA (N-acetyl groups) as well as into compounds derived from pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), hexosephosphates (galactosamine), and pentosephosphates (ribose). The specific radioactities of N-acetylgroups and of the three amino acids were identical. The hexosamine exhibited a two times higher specific radioactivity, and the pentose a 1.6 times higher specific radioactivity than e.g. alanine. M. thermoautotrophicum growing in presence of 3-¹⁴C pyruvate, however, did not incorporate ¹⁴C into cell compounds directly derived from acetyl CoA. Those compounds derived from pyruvate, dicarboxylic acids and hexosephosphates became labelled. The specific radioactivities of alanine, aspartate and glutamate were identical; the hexosamine had a specific radioactivity twice as high as e.g. alanine.The finding that pyruvate was not incorporated into compounds derived from acetyl CoA, whereas acetate was incorporated into derivatives of acetyl CoA and pyruvate in a 1:1 ratio demonstrates that pyruvate is synthesized by reductive carboxylation of acetyl CoA. The data further provide evidence that in this autotrophic CO₂ fixation pathway hexosephosphates and pentosephosphates are synthesized from CO₂ via acetyl CoA and pyruvate.     

Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes

Abstract: CO₂ fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with ¹⁴CO₂ and the incorporation of ¹⁴C into medium or cell extract products was determined. After chromatographic separation of ¹⁴C-labelled products, the fractions of ¹⁴C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the ¹⁴C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO₂ fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [¹⁴C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net ¹⁴CO₂ fixation, but did alter the distribution of ¹⁴C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or ¹⁴C incorporation). Low rates of conversion of α-[¹⁴C]ketoglutarate to [¹⁴C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis.     

Role of carbon dioxide fixation,blood aspartate and glutamate in the adaptation of amphibian brain tissues to a hyperosmotic internal environment

Mechanisms have been examined by which hyperosmotic blood plasma might elevate the levels of aspartate and glutamate in the brain of the toadBufo boreas. CO₂ fixation was assessed by two in vivo methods using [2-¹⁴C]glucose injected intracisternally. Thirty minutes after injection, the¹⁴C labeling of glutamate and aspartate was more than 100 times greater in brain than in liver. In brain tissues, 40+% of¹⁴C atoms appeared to be incorporated into aspartate via the pyruvate carboxylase pathway. Brain tissues of control toads and toads adapting or adapted to hyperosmotic plasma osmolality revealed no differences in the rate of CO₂ fixation as related to glucose utilization or tissue pool sizes of glutamate and aspartate. Elevated levels of these amino acids in blood plasma preceded increases in brain tissues. Carbon atoms required during hyperosmotic adaptation for expansion of amino acid pools in brain tissues may, in part, originate from amino acids in blood but apparently not from CO₂ fixation in brain.     

The Path of Carbon Flow during NO(3)-Induced Photosynthetic Suppression in N-Limited Selenastrum minutum

Nitrate addition to nitrate-limited cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) resulted in a 70% suppression of photosynthetic carbon fixation. In ¹⁴CO₂ pulse/chase experiments nitrate resupply increased radiolabel incorporation into amino and organic acids and decreased radiolabel incorporation into insoluble material. Nitrate resupply increased the concentration of phosphoenolpyruvate and increased the radiolabeling of phosphoenolpyruvate, pyruvate and tricarboxylic acid cycle intermediates, notably citrate, fumarate, and malate. Furthermore, nitrate also increased the pool sizes and radiolabeling of most amino acids, with alanine, aspartate, glutamate, and glutamine showing the largest changes. Nitrate resupply increased the proportion of radiolabel in the C-4 position of malate and increased the ratios of radiolabel in aspartate to phosphoenolpyruvate and in pyruvate to phosphoenolpyruvate, indicative of increased phosphoenolpyruvate carboxylase and pyruvate kinase activities. Analysis of these data showed that the rate of carbon flow through glutamate (10.6 μmoles glutamate per milligram chlorophyll per hour) and the rate of net glutamate production (7.9 μmoles glutamate per milligram chlorophyll per hour) were both greater than the maximum rate of carbon export from the Calvin cycle which could be maintained during steady state photosynthesis. These results are consistent with the hypothesis that nitrogen resupply to nitrogen-limited microalgae results in a transient suppression of photosynthetic carbon fixation due, in part, to the severity of competition for carbon skeletons between the Calvin cycle and nitrogen assimilation (IR Elrifi, DH Turpin 1986 Plant Physiol 81: 273-279).     

Nutrition of Acetobacter rancens S3 and F11 Isolated from Tanks for Vinegar Production

The strains S3 and F11 which were isolated respectively from static and submerged tanks for vinegar production were identified as Acetobacter rancens. Neither strain grew in an ammonium defined medium containing ethanol, glucose, glycerol or organic acids as the sole carbon source. When casamino acids were added, they grew luxuriantly with lactate, ethanol or glycerol as the carbon source and less well with acetate or glucose. They grew, forming much acetic acid, in defined ethanol medium when alanine was supplied in place of casamino acids, but strain S3 showed a longer lag time than strain Fl1. This lag time could be shortened by addition of aspartate and glutamate. These amino acids could be replaced by succinate, fumarate, malate, lactate, pyruvate or propionate but not by glucose. Both strains required lactate or pyruvate in defined glucose medium but many other organic acids, which were effective in defined ethanol medium, were ineffective or slightly effective in glucose medium.     

Carbon Dioxide Fixation by Lupin Root Nodules: II. Studies with C-labeled Glucose, the Pathway of Glucose Catabolism, and the Effects of Some Treatments That Inhibit Nitrogen Fixation

Labeling studies using detached lupin (Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4-¹⁴C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1-¹⁴C]- and [6-¹⁴C]glucose, while with [U-¹⁴C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the “carbon skeletons” for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.     

Metabolism of absorbed aspartate,asparagine, and arginine by rat small intestine in vivo

l-[U-¹⁴C]aspartate, l-[U-¹⁴C]asparagine, and l-[U-¹⁴C]arginine were administered luminally into isolated segments of rat jejunum in situ, and the radioactive products appearing in venous blood from the segment were identified and quantified, in a continuation of similar studies with l-glutamate and l-glutamine (Windmueller H.G. and Spaeth, A. E. (1975) Arch. Biochem. Biophys. 171, 662–672). Aspartate, administered alone (6 mm) or with 18 other amino acids plus glucose, was absorbed more rapidly than glutamate, but, as with glutamate, less than 1% was recovered intact in intestinal venous blood. More than 50% of aspartate carbon was recovered in CO₂, 24% in organic acids, mostly lactate, 12% in other amino acids (alanine, glutamate, proline, ornithine, and citrulline), and 10% in glucose, apparently the first demonstration of gluconeogenesis by intestine in vivo. In contrast to aspartate and glutamine, nearly all asparagine was absorbed intact, less than 1% being catabolized. About 4% of the absorbed dose was incorporated into the acid-insoluble fraction of intestine, as was the case with all the amino acids studied. In conventional or germ-free rats, only 60% of arginine was absorbed intact, while 33% was hydrolyzed to ornithine and urea. The urea and 38% of the ornithine were released into the blood; the remaining ornithine was metabolized further by intestine to citrulline, proline, glutamate, organic acids, and CO₂. Catabolism of several amino acids from the lumen plus glutamine from arterial blood may provide an important energy source in small intestine.     

DECREASED METABOLISM IN VIVO OF GLUCOSE INTO AMINO ACIDS OF THE BRAIN OF THIAMINE-DEFICIENT RATS AFTER TREATMENT WITH PYRITHIAMINE

Abstract— Thiamine deficiency produced by administration of pyrithiamine to rats maintained on a thiamine-deficient diet resulted in a marked disturbance in amino acid and glucose levels of the brain. In the two pyrithiamine-treated groups of rats (Expt. A and Expt. B) there was a significant decrease in the levels of glutamate (23%, 9%) and aspartate (42%, 57%), and an increase in the levels of glycine (26%, 27%) in the brain, irrespective of whether the animals showed signs of paralysis (Expt. A) or not (Expt. B). as a result of thiamine deficiency. A significant decrease in the levels of γ-aminobutyrate (22%) and serine (28%) in the brain was also observed in those pyrithiamine-treated rats which showed signs of paralysis (Expt. A). Threonine content increased by 57% in Expt. A and 40% in Expt. B in the brain of pyrithiamine-treated rats, but these changes were not statistically significant. The utilization of [U-¹⁴C]glucose into amino acids decreased and accumulation of glucose and [U-¹⁴C]glucose increased significantly in the brain after injection of [U-¹⁴C]glucose to pyrithiamine-treated rats which showed abnormal neurological symptoms (Expt. A). The decrease in ¹⁴C-content of amino acids was due to decreased conversion of [U-¹⁴C]glucose into alanine, glutamate, glutamine, aspartate and γ-aminobutyrate. The flux of [¹⁴C]glutamate into glutamine and γ-aminobutyrate also decreased significantly only in the brain of animals paralysed on treatment with pyrithiamine. The decrease in the labelling of, amino acids was attributed to a decrease in the activities of pyruvate dehydrogenase and α-oxoglutarate dehydrogenase in the brain of pyrithiamine-treated rats. The measurement of specific radioactivity of glucose, glucose-6-phosphate and lactate also indicated a decrease in the activities of glycolytic enzymes in the brain of pyrithiamine-treated animals in Expt. A only. It was suggested that an alteration in the rate of oxidation in vivo of pyruvate in the brain of thiamine-deficient rats is controlled by the glycolytic enzymes, probably at the hexokinase level. The lack of neurotoxic effect and absence of significant decrease in the metabolism of [U-¹⁴C]glucose in the brain of pyrithiamine-treated animals in Expt. B were probably due to the fact that animals in Expt. B were older and weighed more than those in Expt. A, both at the start and the termination of the experiments.     

Amino acids in ram testicular fluid and semen and their metabolism by spermatozoa

1. The testis of the ram secretes considerable amounts of amino acids (200μmoles/day) into the fluid collected from the efferent ducts. The principal amino acid in this testicular fluid is glutamate, which is present in concentrations about eight times those in testicular lymph or in blood from the internal spermatic vein. 2. The concentration of glutamate in seminal plasma from the tail of the epididymis is about ten times that in testicular fluid, and, though glutamate is the major amino acid in ejaculated seminal plasma, its concentration is less than in epididymal plasma. 3. After the intravenous infusion of [U-¹⁴C]glucose, labelled glutamate was found in the testicular fluid. Radioactivity was also detected in alanine, glycine, serine plus glutamine and aspartate. Alanine had the highest specific activity, about 50% of the specific activity of blood glucose. 4. When [U-¹⁴C]glutamate was infused, the specific activity of glutamate in testicular fluid was only about 2% that in the blood plasma. 5. Testicular and ejaculated ram spermatozoa oxidized both [U-¹⁴C]glutamate and [U-¹⁴C]leucine to a small extent, but neither substrate altered the respiration from endogenous levels. 6. No radioactivity was detected in testicular spermatozoal protein after incubation with [U-¹⁴C]glutamate or [U-¹⁴C]leucine. Small amounts of radioactivity were detected in protein from ejaculated ram spermatozoa after incubation with [U-¹⁴C]glutamate. 7. The carbon of [U-¹⁴C]glucose was incorporated into amino acids by testicular spermatozoa; most of the radioactivity occurred in glutamate.     

LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the brain.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

The incorporation of acetate by the chemoautotroph Thiobacillus neapolitanus strain C

Summary Cultures of Thiobacillus neapolitanus strain C assimilate ¹⁴C-labelled acetate and aspartate. Both carbon atoms of acetate are incorporated, and 25% of the cell carbon can arise from acetate. Aspartate-¹⁴C contributes 4–5% of the cell carbon, and is found in pyrimidines and in protein as aspartate and its related amino acids. Acetate-¹⁴C contributes to lipid, glutamate, arginine, proline and leucine, but not to aspartate. Acetate assimilation by washed organisms requires carbon dioxide and energy from thiosulphate oxidation. Degradation of ¹⁴C-glutamic acid from acetate-¹⁴C-labelled bacteria; the accumulation of ¹⁴C-citrate in the presence of fluoroacetate and [¹⁴C] acetate; short-term kinetic experiments on acetate-¹⁴C turnover; and the demonstration of citrate synthesis by cell-free extracts all indicate glutamate synthesis from -ketoglutarate formed by reactions of the tricarboxylic acid cycle. The cycle is believed to be incomplete, probably not proceeding further than -ketoglutarate, and functions as a glutamate-synthesising system, using oxaloacetate derived solely from carbon dioxide fixation. Malate synthase (and the glyoxylate cycle) appear to be insignificant in the metabolism, but extracts did form citramalate from acetate and pyruvate.     

Carbohydrate and Amino Acid Metabolism in the Ectomycorrhizal Ascomycete Sphaerosporella brunnea during Glucose Utilization : A C NMR Study

Nuclear magnetic resonance spectroscopy was utilized to study the metabolism of [1-¹³C]glucose in mycelia of the ectomycorrhizal ascomycete Sphaerosporella brunnea. The main purpose was to assess the biochemical pathways for the assimilation of glucose and to identify the compounds accumulated during glucose assimilation. The majority of the ¹³C label was incorporated into mannitol, while glycogen, trehalose and free amino acids were labeled to a much lesser extent. The high enrichment of the C1/C6 position of mannitol indicated that the polyol was formed via a direct route from absorbed glucose. Randomization of the ¹³C label was observed to occur in glucose and trehalose leading to the accumulation of [1,6-¹³C]trehalose and [1,6-¹³C]glucose. This suggests that the majority of the glucose carbon used to form trehalose was cycled through the metabolically active mannitol pool. The proportion of label entering the free amino acids represented 38% of the soluble ¹³C after 6 hours of continuous glucose labeling. Therefore, amino acid biosynthesis is an important sink of assimilated carbon. Carbon-13 was incorporated into [3-¹³C]alanine and [2-¹³C]-, [3-¹³C]-, and [4-¹³C]glutamate and glutamine. From the analysis of the intramolecular ¹³C enrichment of these amino acids, it is concluded that [3-¹³C]pyruvate, arising from [1-¹³C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase, and pyruvate carboxylase (or phosphoenolpyruvate carboxykinase). Intramolecular ¹³C labeling patterns of glutamate and glutamine were similar and are consistent with the operation of the Krebs cycle. There is strong evidence for (a) randomization of the label on C2 and C3 positions of oxaloacetate via malate dehydrogenase and fumarase, and (b) the dual biosynthetic and respiratory role of the citrate synthase, aconitase, and isocitrate dehydrogenase reactions. The high flux of carbon through the carboxylation (presumably pyruvate carboxylase) step indicates that CO₂ fixation is an important component of the carbon metabolism in S. brunnea, and it is likely that this anaplerotic role is particularly prevalent during NH₄⁺ assimilation. The most relevant information resulting from this investigation is (a) the occurrence of the mannitol cycle, (b) a large part of the trehalose pool is synthesized after the cycling of glucose-carbon through the mannitol cycle, and (c) pyruvate (or phosphoenolpyruvate) carboxylation plays an important role in the primary metabolism of glucose-fed mycelia.     

Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D-[1-13C]Glucose Metabolism

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ-aminobutyrate (GABA), glutamine, and aspartate. d -[1^-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for ¹³C-NMR analysis and amino acid analysis. The absolute amount of ¹³C present within each carbon-atom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance ¹³C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from ¹³C-¹³C coupling.     

Effect of glucose on pyruvate utilization by Rhodotorula glutinis

Summary The utilization of glucose and pyruvate by the yeast Rhodotorula glutinis in a medium containing both carbon sources has been studied. Glucose is readily consumed whereas the uptake of pyruvate is completely blocked by the presence of the sugar.The content of pyruvate kinase and phosphoenolpyruvate carboxykinase in R. glutinis cells growing on glucose plus pyruvate are drastically affected with time by the disappearance of the sugar from the culture medium. After complete exhaustion of glucose, the level of pyruvate kinase drops sharply down to a minimum whereas that of phosphoenolpyruvate carboxykinase rises abruptly up to a maximum.Feeding experiments with labelled compounds show that glucose affects the utilization of the amino acids alanine and aspartate, and conversely that the amino acids influence the utilization of the sugar. Glucose breakdown and its incorporation into polysaccharides is controlled by the amino acids and gluconeogenesis from the amino acids is controlled by the sugar.     

Intermediary metabolism in Legionella pneumophila: utilization of amino acids and other compounds as energy sources.

The utilization of amino acids and other compounds as carbon and energy sources by Legionella pneumophila was examined. Based on the stimulation of oxygen consumption in washed-cell suspensions, glutamate, serine, threonine, and tyrosine were the only amino acids which were utilized as energy sources. Other stimulators of oxygen uptake were lactate, pyruvate, acetate, fumarate, and succinate. Citrate was a good stimulator only when the bacteria were grown in the presence of the substrate. Radiolabeling studies showed that [14C]glutamate was rapidly metabolized, with the label distributed evenly in all cell fractions. [14C]pyruvate and [14C]acetate were incorporated into the lipid-containing cell fraction, whereas glucose and glycerol were found in both the lipid- and polysaccharide-containing cell fractions. Radiorespirometry of differentially labeled [14C]glucose indicated that this compound was metabolized primarily by the pentose phosphate and Entner-Doudoroff pathways rather than by the glycolytic pathway.     

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.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.