Glycolysis during gluconeogenesis in cotyledons of Cucurbita pepo

The aim of this work was to investigate the extent of glycolysis during gluconeogenesis in the germination of marrow (Cucurbita pepo L. var. medullosa Alef.). The activities of phosphofructokinase (E.C. 2.7.1.11) in extracts of cotyledons, of seeds, and seedlings grown in the dark for 2, 5, and 8 days were 3·5, 4·8, 9·4, and 11·8 nmol substrate consumed per cotyledon per min, respectively. The comparable figures for pyruvate kinase (E.C. 2.7.1.41) were 16·3, 72·3, 974, and 1485. The patterns of ¹⁴CO₂ production from [1-¹⁴C], [2-¹⁴C], [3,4-¹⁴C], and [6-¹⁴C]glucose indicated that at all the above stages of germination glycolysis was appreciable and predominated over the pentose phosphate pathway. These patterns, and the distribution of label from [1-¹⁴C], and [3-¹⁴C]pyruvate supplied to 5-day-old cotyledons, indicated that the pyruvate formed in glycolysis was converted to acetyl units that were used primarily in biosyntheses. It is concluded that glycolysis occurred at all the stages of germination examined and was particularly active during gluconeogenesis. It is suggested that the significance of this glycolysis is the provision of intermediates for biosyntheses, a need that may not be met by corresponding gluconeogenic intermediates as these may be retained within organelles.     

A new mathematical approach to the metabolism of [14C]glucose, with applications to sensory ganglia of chicken embryos

Abstract— The available models of carbohydrate metabolism are not suitable for analysis of experiments on dorsal root ganglia of chicken embryos because they assume that certain products of the pentose cycle mix freely with those of glycolysis, which appears not to be true in this tissue, and because full isotopic equilibration, needed before the start of measurements, is not achieved while the excised ganglia are reasonably fresh. Therefore, new equations were developed which assume only a steady state of relevant metabolic intermediates and make use of the process of isotopic equilibration as a source of information. It is also assumed that an initially unknown but calculable fraction of the products of each pentose cycle re-enters the next cycle, the remainder leaking either to glycolysis or to the incubation medium. From measurements of the time course of output of labelled CO₂ in the presence of [1-¹⁴C]- and [6-¹⁴C]glucose and the incorporation and release in lactate of labelled C from [l-¹⁴C]glucose, the equations permit the estimation of many features of carbohydrate metabolism, such as the partitioning of material between the pentose cycle and glycolysis, the partitioning of CO₂ output between the pentose and citric acid cycles, the partitioning of the products of glycolysis between CO₂ and other destinations, such as lactate, and the degree of recycling from one pentose cycle into the next. In addition, the time course of labelled CO₂ output from [2-¹⁴C]glucose can be predicted; this, by comparison with the observed output, serves to support some variants of the basic model, while invalidating others. In dorsal root ganglia from 15-day chicken embryos, the assumption of a metabolic steady state was supported by a constant output of labelled CO² from [l-¹⁴C]glucose for 15 or more hours, except for the initial period of isotopic equilibration. By use of the new equations, it is concluded that in these ganglia (a) recycling in the pentose cycle can be 100% efficient in some incubation conditions, but not in others, (b) more CO₂ is released from the pentose cycle than from the citric acid cycle, (c) large, quantifiable differences exist between the utilization of the various carbon atoms of glucose, and (d) a pool of intermediates within the pentose cycle, with a time constant of about 1 h, explains a large delay observed in the output of C-6 of glucose into CO², which occurs with a time constant as long as 5 h under some conditions. Under conditions where recycling is complete in the pentose cycle, this cycle must operate in isolation from glycolysis, which would otherwise convert much of the output of the pentose cycle to lactate. This may explain the role of fructose-1,6-diphospha-tase in the tissue, without recourse to the oft-proposed, puzzling, and ATP-degrading‘futile cycle’between fructose-6-P and fructose-1,6-diP. It is proposed that the new equations may be suitable for similar analyses on some, but not all, other tissues.     

Energy metabolism in hypoxic astrocytes: Protective mechanism of fructose-1,6-bisphosphate

The protective effects of fructose-1,6-biphosphate (FBP) during hypoxia/ischemia are thought to result from uptake and utilization of FBP as a substrate for glycolysis or from stimulation of glucose metabolism. To test these hypotheses, we measumed CO₂ and lactate production from [6-¹⁴C]glucose, [1-¹⁴C]glucose, and [U-¹⁴C]FBP in normoxic and hypoxic cultured astrocytes with and without FBP present. FBP had little effect on CO₂ production by glycolysis, but increased CO₂ production by the pentose phosphate pathway. Labeled FBP produced very small amounts of CO₂. Lactate production from [1-, and 6-¹⁴C]glucose increased similarly during hypoxic hypoxia; the increase was independent of added FBP. Labeled lactate from [U-¹⁴C]FBP was minimal. We conclude that exogenous FBP is not used by astrocytes as a substrate for glycolysis and that FBP alters glucose metabolism.     

Activity of the pentose phosphate pathway during lignification

Summary We did this work to see if there is a correlation between lignin synthesis and the activity of the pentose phosphate pathway. Excision of the third internode of the stem of Coleus blumei Benth. followed by incubation on sucrose and indoleacetic acid led to extensive formation of tracheids. During this lignification we determined the activities of glucose-6-phosphate dehydrogenase and fructose-1,6-diphosphate aldolase, and the extent to which [1-¹⁴C]-,[3,4-¹⁴C]-, and [6-¹⁴C]glucose labelled CO₂ and the major cellular components. The results indicate that the pentose phosphate pathway was active during lignification, and that the activity of this pathway relative to glycolysis increased at the onset of lignification. Explants of storage tissue of Helianthus tuberosus L. were cultured under conditions which caused extensive lignification. ¹⁴CO₂ production from [1-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose indicated activity of the pentose phosphate pathway during tracheid formation. We suggest that lignification is accompanied by appreciable activity of the pentose phosphate pathway and that this could provide the reducing power for lignin synthesis.Abbreviations NADP nicotinamide-adenine dinucleotide phosphate - IAA indoleacetic acid     

The catabolism of glucose in Tenebrio molitor: The effects of corpora cardiaca

During the development of the mealworm Tenebrio molitor, the effects of corpora cardiaca (CC) extracts on glucose catabolism were tested. In control insects and in insects receiving CC extracts, the activity of the pentose cycle and the glycolytic-citric acid cycle, were evaluated in vivo by a radiorespirometric method using [1-¹⁴C] glucose and [6-¹⁴C] glucose as substrates. The CC extracts strongly divert glucose from the pentose phosphate pathway, which is very active in Tenebrio molitor. Glucose oxidation is reduced by the CC extracts in pupae and adults but is increased in last instar larvae. It seems that the effects of CC extracts vary depending upon the state of carbohydrate reserves.     

Carbohydrate oxidation by leucoplasts from suspension cultures of soybean (Glycine max L.)

The aim of this work was to discover how leucoplasts from suspension cultures of soybean (Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with ¹⁴C-labelled substrates led to ¹⁴CO₂ production, that was dependent upon leucoplast intactness, from [U-¹⁴C]glucose 6-phosphate, [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C] fructose 6-phosphate and [U-¹⁴C]glucose+ATP, but not from [U-¹⁴C]fructose-1,6-bisphosphate or [U-¹⁴C]triose phosphate. The yield from [U-¹⁴C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1-¹⁴C]-, [2-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose 6-phosphate were supplied to leucoplasts significant ¹⁴CO₂ production that was dependent upon leucoplast intactness was found only for [1-¹⁴C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.S.A.C. thanks the Science and Engineering Research Council for a research studentship.     

The effect of nitrogen deficiency on the growth and metabolism of Lemna minor L.

When Lemna is deprived of nitrogen, growth and respiration decrease and the pattern of ¹⁴CO₂ release from [1-¹⁴C]glucose and [6-¹⁴C]glucose is consistent with a relatively strong inhibition of glycolysis. Protein degradation is enhanced but the concentration of free amino acid decreases. It is argued that the biological significance of the increased protein degradation does not lie in its contribution to respiration but as a mechanism to replace one set of enzymes adapted to a particular environmental condition (high nitrogen) with another set of enzymes adapted for low nitrogen in the environment. The change in enzyme pattern associated with the change from high to zero nitrogen in the growth medium has been examined for nine enzymes. The changes in activity observed are consistent with the observed apparent inhibition of glycolysis during nitrogen starvation, but do not explain the inhibition of the pentose phosphate pathway.     

Anaerobiosis in Echinochloa crus-galli (Barnyard Grass) Seedlings : Intermediary Metabolism and Ethanol Tolerance

Tolerance to ethanol and the ability to metabolize key intermediary substrates under anaerobiosis were studied in Echinochloa crus-galli (L.) Beauv. var oryzicola seeds to further characterize the mechanisms which enable it to germinate and grow without O₂.

Our results indicate that E. crus-galli var oryzicola possesses an inherently high tolerance to ethanol and is able to metabolize low levels of ethanol in the absence of O₂. Concentrations of ethanol 45-fold greater than endogenous levels did not prove toxic to germinating seeds.

Five-day anaerobically grown seedlings of E. crus-galli var oryzicola metabolized added [¹⁴C]sucrose primarily to CO₂ and ethanol. Of the soluble compounds labeled, the phosphorylated intermediates of glycolysis and the oxidative pentose phosphate pathway predominated more under anaerobiosis than in air. In addition, organic acids and lipids were labeled from [¹⁴C]sucrose, the latter indicating that metabolism of carbohydrate via acetyl-CoA occurred in the absence of O₂. Lipids were also labeled when seeds were supplied with [¹⁴C]ethanol or [¹⁴C]acetate. Labeling experiments using the above compounds plus [¹⁴C]NaHCO₃, showed further labeling of organic acids; succinate and citrate being labeled under nitrogen, while fumarate was formed in air.

The above metabolic characteristics would allow for the maintenance of an active alcoholic fermentation system which, along with high alcohol dehydrogenase activity, would continue to recycle NAD and result in continued energy production without O₂. In addition, Echinochloa's ability to metabolize carbohydrate intermediates and to synthesize lipids indicates that mechanisms exist for providing the carbon intermediates for biosynthesis, particularly membrane synthesis for growth, even in the absence of O₂.

     

The metabolism of galactose and the raffinose oligosaccharides by germinating bambarra groundnut seeds

Stachyose is present in the highest amount in the soluble sugar fraction of dry bambarra groundnut cotyledons, followed in descending order by raffinose, sucrose and verbascose. During germination in the dark, the stachyose and raffinose content decrease rapidly, but there is little change in the relatively small amount of verbascose present. The sucrose content increases rapidly during the first two weeks and decreases thereafter. Free glucose and fructose were present in the cotyledons after the 7th day and gradually increased in amount with time of germination. Free galactose and other galactose-containing oligosaccharides were not detected in either the dry or germinated bambarra seeds. During germination, galactose was the only identifiable sugar, aside from traces of sucrose, glucose and fructose, in the extracted soluble sugar fraction in the embryonic axes of all ages when the tissue was incubated with D-[1¹⁴C] galactose. With the cotyledons, however, most of the radioactivity was in glucose and fructose during the early period of germination and in sucrose later. A small fraction of the radioactivity was lost as CO₂.     

The fate of [14C]glucose during cold-hardening in Eurosta solidaginis (Fitch)

Glucose catabolism in overwintering larvae Eurosta solidaginis was examined to determine the relative contributions of glycolysis and the pentose phosphate pathway to polyol synthesis at different temperatures. Rates of ¹⁴CO₂ evolution were determined after injection of [¹⁴C]1-glucose, [¹⁴C]6-glucose, and [¹⁴C]3,4-glucose. In addition incorporation of label from each isotope into sorbitol and glycerol was monitored. The respirometric studies showed a relative increase in pentose phosphate activity between 10 and 5°C. Similar results were obtained from the changes of radioactivities incorporated into glycerol, although the activation of the pentose phosphate pathway was low. The conversion of [¹⁴C]glucose to glycerol was highest at 10°C, suggesting that maximum glycerol synthesis may occur at this temperature. Radioactivity appeared in the sorbitol fraction of larvae incubated at temperatures below 5°C. Late autumn larvae converted more [¹⁴C]glucose than did early autumn larvae.     

Oxidation of Glucose,Ribose, Alanine,and Glutamate by Leishmania braziliensis panamensis1

The metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose, [1-¹⁴]ribose, [1-¹⁴C]- and [U-¹⁴C]alanine, and [1-¹⁴C]- and [5-¹⁴C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of ¹⁴CO₂ produced from [1-¹⁴C]glucose to that from [6-¹⁴C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of ¹⁴CO₂ from [1-¹⁴C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of ¹⁴CO₂ production from [1-¹⁴C]glucose was almost linear with time of incubation, whereas that of [6-¹⁴C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26°C to 34°C had no appreciable effect on the rates or time courses of oxidation of either [1-¹⁴C]- or [6-¹⁴C]glucose or of [1-¹⁴C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of ¹⁴CO₂ produced from [1-¹⁴C]- to [U-¹⁴C]alanine and from [1-¹⁴C]- to [5^-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide. Heating the cultures for 6 or 12 h at 34°C, which converts the promastigotes into an ellipsoidally shaped intermediate form, decreased the rates of oxidation of glucose, alanine, and glutamate. The oxidation of glutamate decreased by about 50% and 70% after a 6-h or 12-h heat treatment, respectively. Returning the heated cultures to 26°C initiated a reversion to the promastigote form and recovery of the rate of glucose oxidation, but glutamate oxidation did not return to control levels by 19 h at 26°C.     

Pathways of carbohydrate oxidation in leaves of Pisum sativum and Triticum aestivum

The aim of this work was to establish the pathways of carbohydrate oxidation used in the dark by leaves of Pisum sativum and Triticum aestivum. Segments of young and mature leaves of pea released the carbons of glucose-[¹⁴C] as ¹⁴CO₂ in the order 3,4 > 1 > 2 > 6 whereas in segments of young and mature leaves of wheat the order was 3,4 > 1 > 6 > 2. The detailed labelling of the constituents of mature leaves of wheat by glucose-[1-¹⁴C], -[2-¹⁴C], -[3,4-¹⁴C], and -[6-¹⁴C] was determined and showed that the high yield of CO₂ from C-6 relative to that from C-2 was due to release of C-6 during pentan synthesis. Estimates were made of the maximum catalytic activities of phosphofructokinase and glucose-6-phosphate dehydrogenase in pea and wheat leaves of three ages. The results of all the above investigations strongly indicate that both pea and wheat leaves in the dark oxidize carbohydrate via glycolysis and the pentose phosphate pathway with the latter accounting for no more than a third of the total. No evidence was obtained of any major change in the relative activities of the two pathways during the development of either type of leaf.     

Integrated 13C-metabolic flux analysis of 14 parallel labeling experiments in Escherichia coli

The use of parallel labeling experiments for ¹³C metabolic flux analysis (¹³C-MFA) has emerged in recent years as the new gold standard in fluxomics. The methodology has been termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. In this contribution, we have tested the limits of COMPLETE-MFA by demonstrating integrated analysis of 14 parallel labeling experiments with Escherichia coli. An effort on such a massive scale has never been attempted before. In addition to several widely used isotopic tracers such as [1,2-¹³C]glucose and mixtures of [1-¹³C]glucose and [U-¹³C]glucose, four novel tracers were applied in this study: [2,3-¹³C]glucose, [4,5,6-¹³C]glucose, [2,3,4,5,6-¹³C]glucose and a mixture of [1-¹³C]glucose and [4,5,6-¹³C]glucose. This allowed us for the first time to compare the performance of a large number of isotopic tracers. Overall, there was no single best tracer for the entire E. coli metabolic network model. Tracers that produced well-resolved fluxes in the upper part of metabolism (glycolysis and pentose phosphate pathways) showed poor performance for fluxes in the lower part of metabolism (TCA cycle and anaplerotic reactions), and vice versa. The best tracer for upper metabolism was 80% [1-¹³C]glucose+20% [U-¹³C]glucose, while [4,5,6-¹³C]glucose and [5-¹³C]glucose both produced optimal flux resolution in the lower part of metabolism. COMPLETE-MFA improved both flux precision and flux observability, i.e. more independent fluxes were resolved with smaller confidence intervals, especially exchange fluxes. Overall, this study demonstrates that COMPLETE-MFA is a powerful approach for improving flux measurements and that this methodology should be considered in future studies that require very high flux resolution.     

Fluxes of carbohydrate metabolism in ripening bananas

The major fluxes of carbohydrate metabolism were estimated during starch breakdown by ripening bananas (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21° C. Production of CO₂ and the contents of starch, sucrose, glucose and fructose of intact fruit were determined for a period of 10 d that included the climacteric. The detailed distribution of label was determined after supplying the following to cores of pulp from climacteric fruit: [U-¹⁴C]-, [1-¹⁴C]-, [3,4-¹⁴C]-and [6-¹⁴C]glucose, [U-¹⁴C]glycerol, ¹⁴CO₂. The data obtained were used to estimate the following fluxes, values given as mol hexose · (g FW)^–1 · h^–1 in parenthesis: starch to hexose monophosphates (5.9) and vice versa (0.4); hexose monophosphates to sucrose (7.7); sucrose to hexose (4.7); hexose to hexose monophosphate (3.8); glycolysis (0.5–1.6); triose phosphate to hexose monophosphates (0.14); oxidative pentose-phosphate pathway (0.48); CO₂ fixation in the dark (0.005). These estimates are related to our understanding of carbohydrate metabolism during ripening.We both thank Mr Richard Trethewey for his constructive criticism: S.A.H. thanks the Managers of the Broodbank Fund for a fellowship.     

Effect of gibberellic acid on sterol production in Corylus avellana seeds

Gibberellic acid-induced germination of hazel seeds was accompanied by little change in the sterol content of the cotyledons. Dormant and germinating cotyledons rapidly incorporated [2-¹⁴C]MVA into squalene which was slowly converted to sterols. Gibberellin treatment induced an increase in the incorporation of [2-¹⁴C]MVA into cotyledon esterified sterols. An increase in free sterols occurred in the germinating embryonic axes, with increased relative amounts of stigmasterol and campesterol in the free 4-desmethylsterols. Germination was accompanied by increased incorporation of [2-¹⁴C]MVA into free and esterified sterols in the embryonic axes.     

Effects of intestinal insulin-like peptide on glucose catabolism in male adult Locusta migratoria

An insulin-like peptide (ILP) extracted from midgut of 25-day-old male adult Locusta migratoria can modify the relative activity of the two main pathways of glucose catabolism. The effect of ILP on the activity of the pentose cycle and the glycolytic-citric acid cycle in Locusta migratoria was evaluated by a radiorespirometric method by means of [1-¹⁴C] glucose and [6-¹⁴C]glucose as substrates. The time course of the ILP effect was determined. The insulin-like peptide increases the relative activity of the pentose cycle. This effect is rapid and of short duration. Injected mammalian insulin has a similar effect.     

Activity of the Pentose Phosphate and Glycolytic Pathways During Anaerobic Germination of Echinochloa crus-galli (barnyard grass) Seeds

Changes in the activity of key enzymes in glycolysis and theoxidative pentose phosphate pathway were studied in Echinochloacrus-galli (L.) Beauv. var. oryzicola seeds during germinationin air or nitrogen. In addition, the metabolism of specificallylabelled [^I4C]glucose was followed to evaluate the activityof both pathways during anaerobic germination. During the 7 d time period studied there was no difference betweenair and nitrogen in phosphofructokinase activity. Under anaerobicconditions, fructose-1, 6-bisphosphate aldolase increased morethan two-fold in 7 d; whereas in air, it decreased. The activityof the pentose phosphate pathway enzyme, glucose-6-phosphatedehydrogenase, increased under N₂ until day three, when it levelledoff, whilst it continued to increase up to day seven in air. Incubation of Echinochloa seedlings with specifically labelledglucose also resulted in differences between anaerobic- andair-grown seedlings. Labelling of phosphorylated sugars andlipids predominated under N₂; whereas in air, malate and fumaratewere the most heavily labelled compounds. In both air and N₂,there was a greater percentage of label in CO2 from [l-¹⁴C]glucose,while [6-14C] resulted in a greater percentage label in ethanol.These differences were more pronounced under N2, especiallyduring the first 24 h of imbibition, suggesting increased activityof the pentose phosphate pathway. Key words: Echinochloa, Anaerobic metabolism, Oxidative pentose phosphate pathway     

Pathways of carbohydrate oxidation during thermogenesis by the spadix of Arum maculatum.

1. The aims of this work were to discover the pathways of carbohydrate oxidation prior to and during thermogenesis by the club of the spadix of Arum maculatum, and whether there was coarse control of these pathways. 2. 14C02 production from [1-14C]-, [3,4-14C]-, and [6-14C]glucose, the detailed distribution of 14C from [1-14C]- and [6-14C]glucose, and the maximum catalytic activities of phosphofructokinase, fructose-1,6-diphosphate aldolase, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase were determined at different stages in the development of the spadix. The results indicate that in the early stages carbohydrate is oxidized via both the pentose phosphate pathway and glycolysis, and that a shift to glycolysis occurs during development so that just before and during thermogenesis glycolysis predominates almost exclusively. 3. During development the activities of phosphofructokinase and glucose-6-phosphate dehydrogenase per club increased 100- ans during spadix development, and indicated that the onset of rapid glycolysis at thermogenesis is regulated by fine control or availability of substrate.