Glucose metabolism and dimorphism in Mucor.

Mucor racemosus fermented glucose to ethanol, carbon dioxide, and glycerol. When this fungus was grown anaerobically in either the yeast or mycelial form, the catabolism of glucose was very similar. Yeast cells shifted to aerobic conditions maintained a high flux of glucose carbon through the glycolytic and pentose phosphate pathways. Mycelial cells grown aerobically catabolized glucose in a manner consistent with a respiratory metabolism. Although there was no consistent pattern of glucose metabolism in the mycelial form of Mucor, growth in the yeast form consistently was correlated with a high flux of glucose carbon through the catabolic pathways.     

Modulation of glycogen and trehalose levels in Micromonospora echinospora (ATCC 15837)

The growth of Micromonospora echinospora was studied in high and low C/N ratio medium using both batch and continuous culture. Asparagine was consumed rapidly in batch cultures where it served as both a nitrogen and carbon source. Glucose consumption was low suggesting that asparagine functions as the major carbon source under these conditions. The effect of nutrient limitation on the accumulation of storage carbohydrate in batch culture revealed an intimate association between nitrogen limitation and the accumulation of carbonaceous reserves. This study revealed that glycogen constituted the major carbohydrate reserve associated with the onset of sporulation. Intracellular trehalose levels were found to be relatively low and may have been affected by the availability of carbon. Continuous culture studies revealed a correlation between glycogen accumulation and increasing growth rate. It was also found that elevated cellular ATP levels correlated with the increase in glycogen, and reduced glycolytic activity. At the higher growth rates cellular ATP levels were elevated and coincided with reduced activity of the key glycolytic enzyme, phosphofructokinase, suggesting that glycogen can act as a convenient energy reservoir when excess carbon flux dictates.     

Glucose and glycogen utilisation in myocardial ischemia - changes in metabolism and consequences for the myocyte

Experimentally, enhanced glycolytic flux has been shown to confer many benefits to the ischernic heart, including maintenance of membrane activity, inhibition of contracture, reduced arrhythmias, and improved functional recovery. While at moderate low coronary flows, the benefits of glycolysis appear extensive, the controversy arises at very low flow rates, in the absence of flow; or when glycolytic substrate may be present in excess, such that high glucose concentrations with or without insulin overload the cell with deleterious metabolises. Under conditions of total global ischemia' glycogen is the only substrate for glycolytic flux. Glycogenolysis may only be protective until the accumulation of metabolises (lactate, H⁺, NADH, sugar phosphates and Pi ) outweighs the benefit of the ATP produced.The possible deleterious effects associated with increased glycolysis cannot be ignored, and may explain some of the controversial findings reported in the literature. However, an optimal balance between the rate of ATP production and rate of accumulation of metabolises (determined by the glycolytic flux rate and the rate of coronary washout), may ensure optimal recovery. In addition, the effects of glucose utilisation must be distinguished from those of glycogen, differences which may be explained by functional compartmentation within the cell.     

On the mechanism of xylitol-dependent inhibition of glycolysis in Streptococcus sobrinus OMZ 176.

1. The mechanism of xylitol-dependent inhibition of glycolysis in Streptococcus sobrinus OMZ 176 was investigated in aerobically and anaerobically grown cells. 2. Glucose-stimulated glycolysis was followed polarographically, by radio-HPLC-analyses of glycolytic intermediates, by measurement of ATP generated, and spectrophotometric monitoring of extent of NAD(P)+/NADPH-status. 3. Xylitol added to suspensions of S. sobrinus inhibited O2 uptake by approximately 20%, and led to a corresponding decrease in rate of lactate formation in aerobic and anaerobic cells. 4. Xylitol also delayed the onset of the glucose-dependent rapid reduction of NAD(P)+ by approximately 1 min, although the total extent of reduction was not significantly affected compared to control cells. 5. The inhibitory effect of xylitol on glucose dependent ATP synthesis, however, was decreased by 70-80%. 6. Hence the dramatic decrease in glucose-dependent synthesis of ATP may be the direct cause of decreased bacterial growth in the presence of xylitol. 7. A mechanism explaining the observed phenomena is proposed.     

Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge? Kinetics of NAD(+) and NADH pools determined in vivo by 13C NMR

The involvement of nicotinamide adenine nucleotides (NAD(+), NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using (13)C and (31)P NMR to monitor in vivo the kinetics of the pools of NAD(+), NADH, ATP, inorganic phosphate (P(i)), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of (13)C-labeled NAD(+) and NADH. The capacity of L. lactis MG1363 to regenerate NAD(+) was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH(-) deficient strain was constructed by double crossover. Upon supply of glucose, NAD(+) was constant and maximal (approximately 5 mm) in the parent strain (MG1363) but decreased abruptly in the LDH(-) strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH(-) strain and NADH reached high levels (2.5 mm), reflecting severe limitation in regenerating NAD(+). However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mm. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by NADH. The ATP/ADP/P(i) content could play an important role.     

Effects of dinitrophenol and oligomycin on the coupling between anaerobic metabolism and anaerobic sodium transport by the isolated turtle bladder

Dinitrophenol (1 x 10^-5 M) has been found to inhibit anaerobic sodium transport by the isolated urinary bladder of the fresh water turtle. Concurrently, anaerobic glycolysis was stimulated markedly. However, tissue ATP levels diminished only modestly, remaining at approximately 75% of values observed under anaerobic conditions without DNP. The utilization of glucose (from endogenous glycogen) corresponded closely to that predicted from the molar quantities of lactate formed. Thus the glycolytic pathway was completed in the presence of DNP and if ATP were synthesized normally during glycolysis, synthesis should have been increased. On the other hand, the decrease in Na transport should have decreased ATP utilization. Oligomycin did not block sodium transport either aerobically or anaerobically, but ATP concentrations did decrease. When anaerobic glycolysis was blocked by iodoacetate, pyruvate did not sustain sodium transport thus suggesting that no electron acceptors were available in the system. Two explanations are entertained for the anaerobic effect of DNP: (a) Stimulation by DNP of plasma membrane as well as mitochondrial ATPase activity; (b) inhibition of a high energy intermediate derived from glycolytic ATP or from glycolysis per se. The arguments relevant to each possibility are presented in the text. Although definitive resolution is not possible, we believe that the data favor the hypothesis that there was a high energy intermediate in the anaerobic system and that this intermediate, rather than ATP, served as the immediate source of energy for the sodium pump.     

Engineering a central metabolic pathway: glycolysis with no net phosphorylation in an Escherichia coli gap mutant complemented with a plant GapN gene.

A cDNA fragment containing the Pisum sativum GapN gene, which encodes the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, was cloned in a prokaryote expression vector. This construct enabled Escherichia coli strain W3CG, a mutant which lacks the glycolytic phosphorylating G3P dehydrogenase, to grow aerobically on sugars. The functionally complemented mutant exhibited high levels of the catalytically active plant enzyme, which renders 3-phosphoglycerate and NADPH, thus bypassing the first substrate level phosphorylation step of the glycolysis. As expected if such a glycolytic bypass would be operative in vivo, this clone failed to grow anaerobically on sugars in contrast to W3CG clones complemented with phosphorylating glyceraldehyde-3-phosphate dehydrogenases. According to the irreversible catabolic character of the non-phosphorylating reaction, the GapN-complemented clone was unable to grow on gluconeogenic substrates. This metabolic engineering approach demonstrates that a pure catabolic Embden-Meyerhof pathway with no net energy yield is feasible.     

DEVELOPMENTAL CHANGES IN GLYCOLYSIS IN RAT CEREBRAL CORTEX

Abstract— The ATP concentration in infant rat cerebral cortex slices which were incubated aerobically with glucose (5 m m ) as substrate was much higher than in those from the adult. The higher ATP concentration in slices from young rat was also obtained when they were incubated aerobically with pyruvate (10 m m ), dl -lactate (20 m m ) and dl -3-nydroxybutyrate (20 m m ) However, when the slices were incubated anaerobically with glucose, the ATP concentration was very low. Thus, the formation of ATP in the slices from the young rat was thought to be mainly due to their oxidative metabolism, as in those from the adult. The amounts of glycolytic key enzymes in rat cerebral cortex (hexokinase. phosphofructokinase and pyruvate kinase) increased with age. Glycolysis was actually shown to be less active in the cerebral slices from young rats than from the adult. In addition it is known that the tricarboxylic acid cycle enzymes in rat cerebrum also increase with age. Consequently, the activity with respect to ATP formation must be lower in the cerebral cortex slices from young rats than from the adult. The fact that ATP was nevertheless higher in the slices from young rats may be explained by a lower rate of degradation. Developmental increases in the amounts of Na⁺-K⁺-ATPase and Mg²⁺ -ATPase in rat cerebral cortex were greater than those of the glycolytic key enzymes. These are discussed in relation to the observation that the rate of aerobic glycolysis in slices from cerebral cortex of young rats was not increased by d -glutamate (5 m m ) and high potassium (50 m m ).     

Energetics of Anaerobic Sodium Transport by the Fresh Water Turtle Bladder

Certain of the metabolic events associated with anaerobic sodium transport by the isolated bladder of the fresh water turtle have been investigated. The data suggest that energy for this transport arises from glycolysis and that endogenous glycogen was the major and perhaps the sole source of substrate. The rate of anaerobic glycolysis, as determined by lactate formation, correlates well with the rate as determined by glycogen utilization. Using lactate formation as the index of anaerobic glycolysis, a linear relationship was observed between glycolysis and net anaerobic sodium transport. In the absence of sodium transport, glycolysis decreased by approximately 45 per cent. Tissue ATP concentrations were maintained at about the same level under anaerobic as under aerobic conditions. Finally if it is assumed that in the conversion of glycogen to lactate anaerobically, 3 moles of ATP are generated per mole of glucose residue, an average of over 15 equivalents of sodium were transported for every mole of ATP generated.     

Glucose,glycolysis, and neurodegenerative diseases

Prolonged survival of a typical postmitotic neuron hinges on a balance between multiple processes, among these are a sustenance of ATP production and protection against reactive oxygen species. In neuropathological conditions, mitochondrial defects often lead to both a drop in ATP levels, as well as increase reactive oxygen species production from inefficient electron transport processes and NADPH-oxidases activities. The former often resulted in the phenomenon of compensatory aerobic glycolysis. The latter stretches the capacity of the cell's redox buffering capacity, and may lead to damages of key enzymes involved in energy metabolism. Several recent reports have indicated that enhancing glucose availability and uptake, as well as increasing glycolytic flux via pharmacological or genetic manipulation of glycolytic enzymes, could be protective in animal models of several major neurodegenerative diseases, including Parkinson's disease, Huntington's disease, and Amyotrophic lateral sclerosis. Activation of canonical Wnt signaling, which improves disease symptoms in mouse models of Alzheimer's disease also appears to work via an elevation of glycolytic enzymes and enhance glucose metabolism. Here, I discuss these findings and the possible underlying mechanisms of how an increase in glucose uptake and glycolysis could be neuroprotective. Increased glycolytic production of ATP would help alleviate energy deficiency, and ATP's hydrotropic effect may enhance solubility and clearance of toxic aggregates prevalent in many neurodegenerative diseases. Furthermore, channeling of glucose into the Pentose Phosphate Pathway would increase the redox buffering capacity of the cell.     

Effects of nitric oxide donor, isosorbide dinitrate, on energy metabolism of rat reticulocytes

Since nitric oxide (NO) in many cells is involved in energy metabolism, the aim of this study was to evaluate the role of isosorbide dinitrate (ISDN), a NO donor, in energy metabolism of rat reticulocytes, particularly due to their high content of hemoglobin--an effective scavenger of NO. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated in the absence (control) or in the presence of different concentrations of ISDN. ISDN decreased total and coupled oxygen consumption (p<0.05) while increased uncoupled oxygen consumption (p<0.05) in a dose- and time-dependent manner. This was followed by enhancement of glycolysis, as measured by increased glucose consumption and lactate accumulation (p<0.05). Levels of all glycolytic intermediates in the presence of ISDN indicate only stimulation of pyruvate kinase activity. ISDN did not alter the concentration of ATP, while increased ADP and AMP levels (p>0.05). In rat reticulocytes under steady-state conditions, 95.4% of overall energy was produced by oxidative phosphorylation but only 4.6% by glycolysis. Due to a reduced coupled oxygen consumption in the presence of ISDN, ATP production via oxidative phosphorylation was significantly diminished. A simultaneous increase of glycolytic ATP production is not enough to ensure constant ATP production. The calculated mean ATP turnover time was prolonged by 199% in the presence of 1.5 mmol/l ISDN. In conclusion, ISDN a) inhibited total and coupled respiration but enhanced uncoupled respiration, b) stimulated glycolysis, c) decreased ATP production and d) prolonged ATP turnover time in rat reticulocytes. These effects were mediated by NO as the effector molecule.     

Glucose-dependent active ATP depletion by koningic acid kills high-glycolytic cells

Many types of cancer cells depend heavily on glycolysis for energy production even in aerobic conditions. We found that koningic acid (KA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), selectively kills high-glycolytic cells through glucose-dependent active ATP deprivation. Out of seven tumor cell lines tested, growth of six cell lines, which had high glycolytic capacity, was inhibited by KA, whereas three normal cell lines, which had low glycolytic activity, were insensitive to KA. The growth inhibition and caspase-independent cell death in sensitive cells were related to severe ATP depletion that was promoted by glucose phosphorylation. Although GAPDH was completely inhibited in KA-insensitive CHO-K1 cells, KA-mediated ATP depletion was less extensive and transient, possibly due to utilization of ketogenic essential amino acids as energy source. KA suppressed Ehrlich ascites tumor growth in vivo and benefited the survival of the affected mice.     

The bifunctional aldehyde–alcohol dehydrogenase controls ethanol and acetate production in Entamoeba histolytica under aerobic conditions

By applying metabolic control analysis and inhibitor titration we determined the degree of control (flux control coefficient) of pyruvate:ferredoxin oxidoreductase (PFOR) and bifunctional aldehyde–alcohol dehydrogenase (ADHE) over the fluxes of fermentative glycolysis of Entamoeba histolytica subjected to aerobic conditions. The flux-control coefficients towards ethanol and acetate formation determined for PFOR titrated with diphenyleneiodonium were 0.07 and 0.09, whereas for ADHE titrated with disulfiram were 0.33 and ?0.19, respectively. ADHE inhibition induced significant accumulation of glycolytic intermediates and lower ATP content. These results indicate that ADHE exerts significant flux-control on the carbon end-product formation of amoebas subjected to aerobic conditions.     

Pyrophosphate and fructose 2,6-bisphosphate effects on glycolysis in pea seed extracts

The participation of pyrophosphate-dependent phosphofructokinase (PPi-PFK) in plant glycolysis was examined using extracts from pea seeds (Pisum sativum L. cv Alaska). Glycolysis starting with fructose 6-phosphate was measured under aerobic conditions as the accumulation of pyruvate. Pyruvate accumulated in a medium containing PPi and adenosine diphosphate at about two-thirds of the rate in a medium containing adenosine diphosphate and adenosine triphosphate (ATP). The PPi-dependent pyruvate accumulation had the same reactant requirements and sensitivity to glycolysis inhibitors, sodium fluoride, and iodoacetamide, as the well-established ATP-dependent glycolysis. Added fructose 2,6-bisphosphate stimulated both the PPi-dependent pyruvate accumulation and PPi-PFK activity whereas this modulator had no effect on ATP-dependent glycolysis or ATP-PFK. Collectively these results demonstrate a PPi-dependent glycolytic pathway in plants which is responsive to fructose 2,6-bisphosphate.     

Regulation and control of compartmentalized glycolysis in bloodstream formTrypanosoma brucei

Unlike other eukaryotic cells, trypanosomes possess a compartmentalized glycolytic pathway. The conversion of glucose into 3-phosphoglycerate takes place in specialized peroxisomes, called glycosomes. Further conversion of this intermediate into pyruvate occurs in the cytosol. Due to this compartmentation, many regulatory mechanisms operating in other cell types cannot work in trypanosomes. This is reflected by the insensitivity of the glycosomal enzymes to compounds that act as activity regulators in other cell types. Several speculations have been raised about the function of compartmentation of glycolysis in trypanosomes. We calculate that even in a noncompartmentalized trypanosome the flux through glycolysis should not be limited by diffusion. Therefore, the sequestration of glycolytic enzymes in an organelle may not serve to overcome a diffusion limitation. We also search the available data for a possible relation between compartmentation and the distribution of control of the glycolytic flux among the glycolytic enzymes. Under physiological conditions, the rate of glycolytic ATP production in the bloodstream form of the parasite is possibly controlled by the oxygen tension, but not by the glucose concentration. Within the framework of Metabolic Control Analysis, we discuss evidence that glucose transport, although it does not qualify as the sole rate-limiting step, does have a high flux control coefficient. This, however, does not distinguish trypanosomes from other eukaryotic cell types without glycosomes.     

The constitutive K+ pump in Serratia marcescens

Transport of K+ and H+ in the anaeronically and aerobically grown bacterium Serratia marcescens has been studied. The volumes of one cell of the anaerobically and aerobically grown bacterium were 3.7 X 10(-13) cm3 and 2.4 X 10(-13) cm3, respectively. Irrespective of the growth conditions the bacteria manifested the same respiration rate. However, the values of membrane potential for the anaerobically and aerobically grown bacterium were different and equal to -130 mV and -175 mV (interior negative), respectively, in the absence of an exogenic energy source. KCN + DCCD decreases delta psi down to almost zero in both species. DCCD alone decreases delta psi partially in anaerobes and increases delta psi in aerobes, whereas KCN alone reduces delta psi partially in both species. The introduction of glucose into the medium containing K+ reduces the absolute value of delta psi to [-160] mV in aerobes and to [-20] mV in anaerobes. The effect is not observed without external K+. In the presence of arsenate a delta psi is not reduced after the addition of glucose. At pH 7.5-7.8 the ATP level in aerobes grows notably faster than in anaerobes. The H+ extrusion becomes intensified when K+ uptake is activated by the increase in external osmotic pressure. Apparent Km and Vmax for K+ accumulation are 1.2 mM and 0.4 mM.min-1.g-1. The decrease of delta psi by glucose or KCN + DCCD have no effect on the K+ uptake whereas CCCP inhibits potassium accumulation. At the same time, arsenate stabilizes the delta psi value, but blocks K+ uptake. The accumulation of K+ correlates with the potassium equilibrium potential of -200 mV calculated according to the Nernst equation, whereas the delta psi measured was not more than [-25] mV. The calculated H+/ATP stoichiometry was 3.3 for aerobes. It was assumed that a constitutive K+ pump having a K+/ATP ratio equal to 2 or 3 operates in S. marcescens membranes.     

Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions.

Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but much more important was the ability of uncoupling between anabolic biomass formation and catabolic energy substrate consumption. When ammonium started to limit the amount of biomass formed and hence the anabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucose consumption. The primary response when glucose was present in excess of the minimum requirements for biomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, at D = 0.07 h-1, was more than doubled when an additional nitrogen limitation was imposed on the cells compared with that during single glucose limitation. However, the maximum respiratory capacity decreased with decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreased from 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogen concentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation, was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 h-1 obtained under single glucose limitation.     

Kinetic control of ethanol production by Zymomonas mobilis

Summary Zymomonas mobilis UQM 2716 was grown anaerobically in continuous culture (D = 0.1/h; 30° C) 3nder glucose or nitrogen limitation at pH 6.5 or 4.0. The rates of glucose consumption and ethanol production were lowest during glucose-limited growth at pH 6.5, but increased during growth at pH 4.0 or under nitrogen limitation, and were highest during nitrogen-limited growth at pH 4.0. The uncoupling agent CCCP substantially increased the rate of glucose consumption by glucose-limited cultures at pH 6.5, but had much less effect at pH 4.0. Washed cells also metabolised glucose rapidly, irrespective of the conditions under which the original cultures were grown, and the rates were variably increased by low pH and CCCP. Broken cells exhibited substantial ATPase activity, which was increased by growth at low pH. It was concluded that the fermentation rates of cultures growing under glucose or nitrogen limitation at pH 6.5, or under glucose limitation at pH 4.0, are determined by the rate at which energy is dissipated by various cellular activities (including growth, ATP-dependent proton extrusion for maintenance of the protonmotive force and the intracellular pH, and an essentially constitutive ATP-wasting reaction that only operates in the presence of excess glucose). During growth under nitrogen limitation at pH 4.0 the rate of energy dissipation is sufficiently high for the fermentation rate to be determined by the inherent catalytic activity of the catabolic pathway.Abbreviations CCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - qG rate of glucose consumption (g glucose/g dry wt cells/h) - qE rate of ethanol production (g ethanol/g dry wt cells/h) - Y growth yield (g dry wt cells/g glucose) - D dilution rate Offprint requests to: C. W. Jones     

Effects of exogenous donor of nitric oxide-sodium nitroprusside on energy production of rat reticulocytes

The effects of the sodium nitroprusside (SNP), a nitric oxide (NO) donor clinically used in the treatment of hypertensive emergencies on the energy production of rat reticulocytes were investigated. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated without (control) or in the presence of different concentrations of SNP (0.1, 0.25, 0.5, 1.0 mM). SNP decreased total and coupled, but increased uncoupled oxygen consumption. This was accompanied by the stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation. Levels of all glycolytic intermediates indicate stimulation of hexokinase-phosphofructo kinase (HK-PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPD) and pyruvate kinase (PK) activities in the presence of SNP. Due to the decrease of coupled oxygen consumption in the presence of SNP, ATP production via oxidative phosphorylation was significantly diminished. Simultaneous increase of glycolytic ATP production was not enough to provide constant ATP production. In addition, SNP significantly decreased ATP level, which was accompanied with increased ADP and AMP levels. However, the level of total adenine nucleotides was significantly lower, which was the consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level). ATP/ADP ratio and adenylate energy charge level were significantly decreased. In conclusion, SNP induced inhibition of oxidative phosphorylation, stimulation of glycolysis, but depletion of total energy production in rat reticulocytes. These alterations were accompanied with instability of energy status.