Glycolytic flux is conditionally correlated with ATP concentration in Saccharomyces cerevisiae: a chemostat study under carbon- or nitrogen-limiting conditions.

Anaerobic and aerobic chemostat cultures of Saccharomyces cerevisiae were performed at a constant dilution rate of 0.10 h(-1). The glucose concentration was kept constant, whereas the nitrogen concentration was gradually decreasing; i.e., the conditions were changed from glucose and energy limitation to nitrogen limitation and energy excess. This experimental setup enabled the glycolytic rate to be separated from the growth rate. There was an extensive uncoupling between anabolic energy requirements and catabolic energy production when the energy source was present in excess both aerobically and anaerobically. To increase the catabolic activity even further, experiments were carried out in the presence of 5 mM acetic acid or benzoic acid. However, there was almost no effect with acetate addition, whereas both respiratory (aerobically) and fermentative activities were elevated in the presence of benzoic acid. There was a strong negative correlation between glycolytic flux and intracellular ATP content; i.e., the higher the ATP content, the lower the rate of glycolysis. No correlation could be found with the other nucleotides tested (ADP, GTP, and UTP) or with the ATP/ADP ratio. Furthermore, a higher rate of glycolysis was not accompanied by an increasing level of glycolytic enzymes. On the contrary, the glycolytic enzymes decreased with increasing flux. The most pronounced reduction was obtained for HXK2 and ENO1. There was also a correlation between the extent of carbohydrate accumulation and glycolytic flux. A high accumulation was obtained at low glycolytic rates under glucose limitation, whereas nitrogen limitation during conditions of excess carbon and energy resulted in more or less complete depletion of intracellular storage carbohydrates irrespective of anaerobic or aerobic conditions. However, there was one difference in that glycogen dominated anaerobically whereas under aerobic conditions, trehalose was the major carbohydrate accumulated. Possible mechanisms which may explain the strong correlation between glycolytic flux, storage carbohydrate accumulation, and ATP concentrations are discussed.     

Synaptosomal bioenergetics. The role of glycolysis, pyruvate oxidation and responses to hypoglycaemia

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea-pig cerebral cortex is characterized. Essentially all synaptosomes contain functioning mitochondria. There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration. Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose. In glucose-free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios. In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide. Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes. The in-situ mitochondrial membrane potential (delta psi m) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20-mV depolarization, while added pyruvate causes a 6-mV hyperpolarization even in the presence of glucose. Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by alpha-cyano-4-hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted. It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.     

Theoretical phosphorylation rates after addition of a small amount of glucose to intact ascites tumor cells

Changes were measured in glycolytic and respiratory rates during the entire period of glycolysis and respiratory inhibition after addition of 0.08 or 0.15 mM glucose to Ehrlich ascites carcinoma cells in 54 mM phosphate buffer (pH 7.3) at 37°. Glycolytic products fully accounted for the glucose utilized.

Theoretical rates of glycolytic ATP synthesis were calculated from the rates of accumulation of glycolytic products, and rates of oxidative phosphorylation were calculated from respiratory rates, assuming a P:O ratio of 3.0. The maximum in the glycolytic phosphorylation rate curve preceded the minimum in the respiratory phosphorylation rate curve. As a consequence, the total phosphorylation rate curve was biphasic, first rising above, then falling below, and finally returning to the initial, pre-glucose rate. The area under the early rise approximately equalled the area above the later dip and corresponded to between 1 and 2 μmoles of ATP/ml cells. The low rate of change in the ATP content of the cells indicated that most of the change in phosphorylation rate represented changes in both ATP synthesis and ATP utilization.

It is hypothesized that ATP synthesized by glycolysis is more readily available to the ATP-utilizing systems. On addition of glucose, ATP is shifted from a respiratory to a glycolytic reservoir and a period of more rapid ATP utilization associated with a decrease in the level of endogenous substrates involved in the ATP-utilizing reactions ensues; after cessation of glycolysis, the process is reversed, and ATP utilization is slowed for a period while the endogenous substrates increase again.     

The relation between the action potential duration,the increase in resting tension,and ATP content during metabolic inhibition in guinea pig ventricular muscles

To investigate whether the action potential duration (APD) or resting tension was dependent on global ATP content, and whether they were preferentially dependent on glycolytic ATP, APD and resting tension were measured under various metabolic inhibition with corresponding measurement of ATP content in guinea pig ventricular muscles. Oxidative phosphorylation was inhibited by either hypoxic perfusion, the perfusion of sodium cyanide, or 2,4-dinitrophenol. Glycolysis was blocked by the perfusion of iodoacetic acid, and hypoxia with variable glycolytic activities was achieved by hypoxic perfusion in the presence of glucose (5, 10, and 50 mM). APD began to decrease when ATP content decreased to less than 3 mM/kg w.w. from the control level of 4.35 mM/kg w.w. APD shortened significantly and resting tension increased steeply, when ATP content decreased below 1 mM/kg w.w. The dependence of APD and the increase in resting tension on ATP content was not affected by the mode of metabolic block, that is, the inhibition of glycolysis and/or oxidative phosphorylation. Though other factors can affect APD and resting tension, we found no evidence of functional ATP compartmentation, with respect to APD and the increase in resting tension during metabolic inhibition.     

Glycolytic enzymes and a GLUT-1 glucose transporter in the outer segments of rod and cone photoreceptor cells

The presence of glycolytic enzymes and a GLUT-1-type glucose transporter in rod and cone outer segments was determined by enzyme activity assays, glucose uptake measurements, Western blotting, and immunofluorescence microscopy. Enzyme activities of six glycolytic enzymes including hexokinase, phosphofructokinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and lactate dehydrogenase, were found to be present in purified rod outer segment (ROS) preparations. Immunofluorescence microscopy of bovine and chicken retina sections labeled with monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenase have confirmed that these enzymes are present in rod and cone outer segments and not simply contaminants from the inner segments or other cells. Rod outer segments were also found to contain glucose transport activity as detected by 3-O-[14C]methylglucose uptake and exchange. The glucose transporter had a Km of 6.3 mM and a Vmax of 0.15 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for net uptake and a Km of 29 mM and a Vmax of 1.06 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for equilibrium exchange. These Km values for net uptake and equilibrium exchange are similar to values obtained for human red blood cells and are characteristic of GLUT-1-type glucose transporter. The transport was inhibited by both cytochalasin B and phloretin. Western blot analysis and immunofluorescence microscopy using type-specific glucose transporter antibodies indicated that both rod and cone outer segment plasma membranes have a GLUT-1 glucose transporter of Mr 45K as found in red blood cells and brain microsomal membranes. Solid-phase radioimmune competitive inhibition studies indicated that rod outer segment plasma membranes contained 15% the number of glucose transporters found in human red blood cell membranes and had an estimated density of 400 glucose transporter per micron2 of plasma membrane. These studies support the view that outer segments can generate energy in the form of ATP and GTP by anaerobic glycolysis to supply at least some of the energy requirements for phototransduction and other metabolic processes.     

Probing glycolytic and membrane potential oscillations in Saccharomyces cerevisiae

We have investigated glycolytic oscillations under semi-anaerobic conditions in Saccharomyces cerevisiae by means of NADH fluorescence, measurements of intracellular glucose concentration, and mitochondrial membrane potential. The glucose concentration was measured using an optical nanosensor, while mitochondrial membrane potential was measured using the fluorescent dye DiOC 2(3). The results show that, as opposed to NADH and other intermediates in glycolysis, intracellular glucose is not oscillating. Furthermore, oscillations in NADH and membrane potential are inhibited by the ATP/ADP antiporter inhibitor atractyloside and high concentrations of the ATPase inhibitor N, N'-dicyclohexylcarbodiimide, suggesting that there is a strong coupling between oscillations in mitochondrial membrane potential and oscillations in NADH mediated by the ATP/ADP antiporter and possibly also other respiratory components.     

Selection for rapid uptake of scarce or fluctuating resource explains vulnerability of glycolysis to imbalance

Glycolysis is a conserved central pathway in energy metabolism that converts glucose to pyruvate with net production of two ATP molecules. Because ATP is produced only in the lower part of glycolysis (LG), preceded by an initial investment of ATP in the upper glycolysis (UG), achieving robust start-up of the pathway upon activation presents a challenge: a sudden increase in glucose concentration can throw a cell into a self-sustaining imbalanced state in which UG outpaces LG, glycolytic intermediates accumulate and the cell is unable to maintain high ATP concentration needed to support cellular functions. Such metabolic imbalance can result in “substrate-accelerated death”, a phenomenon observed in prokaryotes and eukaryotes when cells are exposed to an excess of substrate that previously limited growth. Here, we address why evolution has apparently not eliminated such a costly vulnerability and propose that it is a manifestation of an evolutionary trade-off, whereby the glycolysis pathway is adapted to quickly secure scarce or fluctuating resource at the expense of vulnerability in an environment with ample resource. To corroborate this idea, we perform individual-based eco-evolutionary simulations of a simplified yeast glycolysis pathway consisting of UG, LG, phosphate transport between a vacuole and a cytosol, and a general ATP demand reaction. The pathway is evolved in constant or fluctuating resource environments by allowing mutations that affect the (maximum) reaction rate constants, reflecting changing expression levels of different glycolytic enzymes. We demonstrate that under limited constant resource, populations evolve to a genotype that exhibits balanced dynamics in the environment it evolved in, but strongly imbalanced dynamics under ample resource conditions. Furthermore, when resource availability is fluctuating, imbalanced dynamics confers a fitness advantage over balanced dynamics: when glucose is abundant, imbalanced pathways can quickly accumulate the glycolytic intermediate FBP as intracellular storage that is used during periods of starvation to maintain high ATP concentration needed for growth. Our model further predicts that in fluctuating environments, competition for glucose can result in stable coexistence of balanced and imbalanced cells, as well as repeated cycles of population crashes and recoveries that depend on such polymorphism. Overall, we demonstrate the importance of ecological and evolutionary arguments for understanding seemingly maladaptive aspects of cellular metabolism.     

The effect of dissolved oxygen tension on the energy metabolism of yeasts]

The specific ATP generation rate in yeasts was examined on the glycolytic pathway and on the respiratory chain as a function of the dissolved oxygen tension of the culture medium. Two different strains were used: Saccharomyces cerevisiae sensitive to the glucose effect and Kluyveromyces fragilis insensitive to the catabolite respression when growing on lactose. The oxidative ATP generation rate followed by these two strains a Michaelis Menten kinetics against the dissolved oxygen concentration. Dissolved oxygen tension only influenced the glycolytic ATP generation rate in Kluyveromyces fragilis. Thus glucose and Pasteur effects are two mutually exclusive regulatory mechanisms of the energy yielding metabolism of the yeasts.     

Control of pyruvate kinase activity during glycolysis and gluconeogenesis in Propionibacterium shermanii

The concentrations of glycolytic intermediates and ATP and the activities of certain glycolytic and gluconeogenic enzymes were determined in Propionibacterium shermanii cultures grown on a fully defined medium with glucose, glycerol or lactate as energy source. On all three energy sources, enzyme activities were similar and pyruvate kinase was considerably more active than the gluconeogenic enzyme pyruvate, orthophosphate dikinase, indicating the need for regulation of pyruvate kinase activity. The intracellular concentration of glucose 6-phosphate, a specific activator of pyruvate kinase in this organism, changed markedly according to both the nature and the concentration of the growth substrate: the concentration (7-10 mM) during growth with excess glucose or glycerol was higher than that (1-2 mM) during growth with lactate or at growth-limiting concentrations of glycerol or glucose. Other glycolytic intermediates, apart from pyruvate, were present at concentrations below 2 mM. Glucose 6-phosphate overcame inhibition of pyruvate kinase activity by ATP and inorganic phosphate. With 1 mM-ATP and more than 10 mM inorganic phosphate, a change in glucose 6-phosphate concentration from 1-2 mM was sufficient to switch pyruvate kinase from a strongly inhibited to a fully active state. The results provide a plausible mechanism for the regulation of glycolysis and gluconeogenesis in P. shermanii.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig.

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase. The calculated cytosolic NAD+/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase.The calculated cytosolic NAD⁺/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase and hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Influence of light and calcium on guanosine 5'-triphosphate in isolated frog rod outer segments.

Frog rod outer segments contain approximately 0.25 mol of GTP and 0.25 mol of ATP per mol of rhodopsin 3 min after their isolation from the retina. UTP and CTP are present at 10-fold and 100-fold lower levels, respectively. Concentrations of GTP and ATP decline in parallel over the next 4 min to reach relatively stable levels of 0.1 mol per mol of rhodopsin. Illumination reduces the concentration of endogenous GTP but not ATP. This light-induced decrease in GTP can be as large as 70% and has a half-time of 7 s. GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity, but partially returns to its dark-adapted level after brief illumination. The magnitude of the decrease increases as a linear function of the logarithm of continuous light intensity at levels which bleach between 5 X 10(2) and 5 X 10(6) rhodopsin molecules/outer segment per second. This exceeds the range of intensities over which illumination causes decreases in the cyclic GMP content and permeability of isolated outer segments (Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). Thus, over 4 log units of light intensity, a sensitivity control mechanism functions to make extended illumination less effective in stimulating a GTP decrease. GTP levels in dark-adapted outer segments are sensitive to changes in calcium concentration in the suspending medium. If the external calcium concentration is reduced to 10(-8) M, GTP concentration is lowered to the same level caused by saturating illumination, and the GTP remaining is no longer light-sensitive. Lowering calcium concentration to intermediate levels between 10(-6) and 10(-8) M reduces GTP to stable intermediate levels, and the GTP remaining can be reduced by light. Restoration of millimolar calcium drives synthesis of GTP, but not of ATP, and GTP lability towards illumination is again observed. These calcium-induced changes in GTP are diminished by the addition of the divalent cation ionophore A23187. Lowering or raising magnesium levels does not influence the GTP concentration. These data raise the possibility that light activates either a calcium transport mechanism driven by the hydrolysis of GTP, or some other calcium-sensitive GTPase activity of unknown function. Known light-dependent reactions involving cyclic nucleotide transformations and rhodopsin phosphorylation appear to account for only a small fraction of the light-induced GTP decrease.     

Regulation of glycolytic flux in an energetically controlled cell-free system: the effects of adenine nucleotide ratios, inorganic phosphate, pH, and citrate

A soluble extract from rat skeletal muscles has been used with purified mitochondrial ATPase (F₁) to develop steady states with respect to glycolytic flux, the concentrations of glycolytic intermediates and inorganic phosphate, and the concentrations and ratios of adenine nucleotides. Incubations were carried out in media resembling the ionic composition in the cell cytoplasm, in an attempt to evaluate the quantitative contributions of various effectors to the overall control mechanism under simulated in vivo conditions. The primary control reaction of glycolytic flux under the conditions studied could be identified with phosphofructokinase, followed by secondary control of the reaction catalyzed by hexokinase. Glycolytic flux was increased with increasing pH over the range 6.6–7.6, both in the absence and presence of ATPase. Without other added effectors, the glycolyzing extract maintained an ATP/ADP ratio of about 50 in the pH range 7.0–7.6, and phosphofructokinase was incompletely suppressed. Addition of increasing amounts of ATPase markedly stimulated glycolytic flux coincident with lowered steady-state ATP/ADP ratios, and decreased accumulation of hexose monophosphates. Control of flux by the ATP/ADP ratio (and simultaneously altered AMP concentration) was less effective if pH (7.3 to 7.6) or phosphate concentration (2 to 20 mm) was increased. Flux through phosphofructokinase was controlled principally when the ATP/ADP ratios were varied in the range between > 50 and 15. The inhibitory effect of citrate was evaluated. Suppression of glycolytic flux and accumulation of hexose monophosphates were dependent on incubation conditions. If the pH was 7.3 or less, and the phosphate concentration low (2 mm), flux through phosphofructokinase was significantly suppressed even at citrate concentrations less than 50 μm. Simultaneous decrease in the steady-state ATP/ADP ratio and elevation of AMP was ineffective in reversing this inhibition. At higher pH and, more dramatically, when the phosphate concentration was increased, sensitivity to citrate inhibition was markedly diminished. These data, taken together with studies of respiratory control with isolated mitochondria (21., 24.), J. Biol. Chem.250, 2275–2282) strongly suggest that adenine nucleotide control of both glycolysis and respiration is exerted when the ratio of free nucleotides (not protein bound) in the cytosol is in the range of 15 to > 50. The data further suggest that citrate plays an important role in the regulation of glycolysis in muscle when the ATP/ADP ratio is high (and the phosphate concentration is correspondingly low), but that this inhibition is overcome by liberation of inorganic phosphate during muscle contraction.     

Effects of insulin and work on fructose 2,6-bisphosphate content and phosphofructokinase activity in perfused rat hearts

The effects of insulin and increased cardiac work on glycolytic rate, metabolite content, and fructose 2,6-bisphosphate (Fru-2,6-P2) content were studied in isolated perfused rat hearts. Steady-state rates of glycolysis increased 5-fold with the addition of insulin to the perfusate or by increasing cardiac pressure-volume work and correlated well in most conditions with changes in substrate concentration (Fru-6-P) and with concentration of the activator, Fru-2,6-P2. There was no correlation with changes in other well known regulators including citrate, ATP, AMP, Pi, or cytosolic phosphorylation potential. Using phosphofructokinase purified from hearts perfused under identical conditions, allosteric kinetic experiments were performed using the metabolite and effector concentrations determined from in vivo experiments. Reaction rates for phosphofructokinase calculated in vitro agreed well with the glycolytic rates measured in vivo and correlated with changes in Fru-6-P but not with other effectors. However, higher Fru-2,6-P2 levels were more effective in maintaining phosphofructokinase activity at high ATP and citrate levels. Kinetic experiments did not indicate a covalent modification of phosphofructokinase. These data indicate that control of cardiac phosphofructokinase and glycolysis may be accomplished by changes in the availability of substrate, Fru-6-P, and activator, Fru-2,6-P2, rather than by citrate, adenine nucleotides, or cytosolic phosphorylation potential as previously suggested.     

Interaction between Chloroplasts and Mitochondria in Microalgae: Role of Glycolysis

In a mutant strain of Chlamydomonas reinhardtii devoid of active ribulose 1,5-bisphosphate carboxylase oxygenase, the addition of mitochondrial inhibitors in the dark resulted in a pronounced decrease in cellular ATP, a fall of the glucose 6-phosphate content, and a rise of the NADPH concentration. These biochemical changes were accompanied by an increase of the fluorescence level, showing changes in the redox state of the chloroplastic electron transport chain. Similar results were obtained in presence of an uncoupler. These data indicated that alterations in the mitochondrial electron transport chain in dark could affect the chloroplastic chain, probably through variations of the glycolysis activity. When mitochondrial oxidases were blocked, illumination of the algae reversed the effect of the inhibitors on the ATP and glucose 6-phosphate concentrations. This last result suggested that the chloroplastic photophosphorylations in these algae played a major role in the control of the glycolytic flux.     

Glycolytic and oxidative metabolism in relation to retinal function

Measurements of lactate production and ATP concentration in superfused rat retinas were compared with extracellular photoreceptor potentials (Fast PIII). The effect of glucose concentration, oxygen tension, metabolic inhibition, and light were studied. Optimal conditions were achieved with 5-20 mM glucose and oxygen. The isolated retina had a high rate of lactate production and maintained the ATP content of a freshly excised retina, and Fast PIII potentials were similar to in vivo recordings. Small (less than 10%) decreases in aerobic and anaerobic lactate production were observed after illumination of dark-adapted retinas. There were no significant differences in ATP content in dark- and light-adapted retinas. In glucose-free medium, lactate production ceased, and the amplitude of Fast PIII and the level of ATP declined, but the rates of decline were slower in oxygen than in nitrogen. ATP levels were reduced and the amplitude of Fast PIII decreased when respiration was inhibited, and these changes were dependent on glucose concentration. Neither glycolysis alone nor Krebs cycle activity alone maintained the superfused rat retina at an optimal level. Retinal lactate production and utilization of ATP were inhibited by ouabain. Mannose but not galactose or fructose produced lactate and maintained ATP content and Fast PIII. Iodoacetate blocked lactate production and Fast PIII and depleted the retina of ATP. Pyruvate, lactate, and glutamine maintained ATP content and Fast PIII reasonably well (greater than 50%) in the absence of glucose, even in the presence of iodoacetate. addition of glucose, mannose, or 2-deoxyglucose to medium containing pyruvate and iodoacetate abolished Fast PIII and depleted the retina of its ATP. It is suggested that the deleterious effects of these three sugars depend upon their cellular uptake and phosphorylation during the blockade of glycolysis by iodoacetate.     

EFFECTS OF LIGHT ON CYCLIC GMP METABOLISM IN RETINAL PHOTORECEPTORS

Abstract— Guanylate cyclase activity of dark-adapted bovine rod outer segments demonstrates a biphasic pattern upon exposure to light. By 10 s of illumination, activity is 20% lower than that observed in dark-adapted outer segments. Activity subsequently increases and then slowly declines to two-thirds of the original activity after 10 min of illumination. In the presence of GTP or ATP, hydrolysis of cyclic GMP is rapidly enhanced by exposure of outer segments to light; the magnitude of this effect is dependent on the amount of substrate present. The rapid effects of light on synthesis and degradation of cyclic GMP indicate that these reactions may be involved in the visual process. The concentration of guanosine 3':5'-cyclic monophosphate (cyclic GMP) is extraordinarily high in dark-adapted bovine rod outer segments and is at least 100-fold that of adenosine 3':5'-cyclic monophosphate (cyclic AMP). No significant decrease in the level of cyclic GMP or cyclic AMP was observed however upon exposure of dark-adapted outer segments to light.     

Adenosine phosphates and the control of glycolysis and gluconeogenesis in yeast

1. Changes in dry weight, protein, RNA and DNA were measured in yeast during adaptation to glycolytic metabolism. 2. Only RNA increased significantly during the lag phase, but during the exponential phase all these cellular components increased in parallel. 3. The concentrations of ATP, ADP, AMP and glucose 6-phosphate were measured in respiring yeast and during the transition to glycolytic metabolism. 4. In respiring cells the concentration of AMP was at its highest and that of ATP was at its lowest; this relationship was reversed in glycolysing cells. 5. ADP concentration was similar in respiring and glycolysing cells, but glucose 6-phosphate concentration was much higher in the glycolysing cells. 6. A possible reason for mitochondrial repression is suggested. 7. It is concluded that adenosine phosphates do not control the direction of glycolytic flux in yeast and an alternative control of glycolysis and gluconeogenesis by enzyme activation and inactivation is suggested.     

The ATP paradox is the expression of an economizing fuel mechanism

The strong negative correlation between glycolytic flux and intracellular ATP concentration observed in yeast has long been an intriguing and counterintuitive phenomenon, which has been referred to as the ATP paradox. Herein, using principles of irreversible thermodynamics it was shown that if the ATP-consuming pathways are more sensitive to extracellular glucose than glycolysis, then upon glucose addition glycolysis performance can switch from an efficient working regime to a dissipative regime, and vice versa, depending on glucose availability. The efficient regime represents a good compromise between high output power and low dissipation, whereas the dissipative working regime offers a higher output power although at a high glucose cost. The physiological and evolutionary implications of this switch strategy are discussed.     

The effect of ATP, GTP and cAMP on the cGMP-dependent conductance of the fragments from frog rod plasma membrane

Using a 'patch-clamp' method in the 'inside-out' configuration, ATP, ADP, AMP-PCP and AMP-PNP have been shown to increase the cGMP-dependent component of the rod plasma membrane conductance 2-4-fold and GTP, GDP but not GMP or nonhydrolyzable GTP analogs GMP-PNP and GTP-gamma-S to abolish the ATP action. The ATP and GTP effects were observed at [EDTA] = 1 mM when magnesium and calcium ions were absent. In about half of the experiments the cGMP-dependent conductance was shown to be increased by cAMP in the micromolar concentration range by 10-50%, the cAMP action did not depend on the presence of nucleoside triphosphates. In vivo ATP, GTP and cAMP are assumed to modulate the sensitivity of the photoreceptor plasma membrane to cGMP.