Regulation of anaerobic glycolysis in Ehrlich ascites tumour cells.

1) In intact Ehrlich ascites tumour cells the anaerobic glycolytic flux rate and pattern of intermediates have been investigated at different pH values of the extracellular medium. 2) As predicted from the dependence of the lactic acid dehydrogenase equilibrium on pH a strong negative correlation between log ([lactate]/[pyruvate]) and pH has been found. 3) The steady state fluxes of glycolysis at pH 8.0 and 7.4 are rather equal, despite significant differences in the intracellular concentrations of glycolytic intermediates. At pH 8.0 the concentrations of ATP, glucose 6-phosphate, and fructose 6-phosphate are lower, and the concentrations of ADP, AMP, fructose 1,6-bisphosphate, triose phosphates, phosphoglycerates, and phosphoenolpyruvate are higher than at pH 7.4. 4) From the analysis of the pH dependent changes of metabolites it follows that different mechanisms are responsible for maintaining equal actual activities of hexokinase, phosphofructokinase and pyruvate kinase at pH 7.4 and 8.0. 5) From an application of the linear theory of enzymatic chains and a calculation of the control strength of the regulatory important enzymes results that hexokinase is evidently rate-limiting for glycolysis, and phosphofructokinase is also significantly influencing the glycolytic flux. Pyruvate kinase and glyceraldehyde phosphate dehydrogenase, on the other hand, do not significantly affect the rate of the overall glycolytic flux in ascites.     

CONTROL OF AEROBIC GLYCOLYSIS AND PYRUVATE KINASE ACTIVITY IN CEREBRAL CORTEX SLICES

Abstract— —The concentrations of glycolytic intermediates were measured in aerobically incubated guinea pig cerebral cortex slices. Increasing the concentration of potassium in the medium increased fructose diphosphate ten-fold and triose phosphates three-fold. Omitting calcium increased all the glycolytic intermediates except pyruvate; triose phosphates were increased most. The changed patterns of the glycolytic intermediate profile in the slices are consistent with the previously proposed hypothesis that the phosphofructokinase is a main regulatory step in glycolysis. Glycolysis is also limited at the step of pyruvate kinase, which is inhibited in cerebral cortex slices. Calcium in the tissue and cellular organization of the slices were shown to be responsible for this inhibition. It was concluded that the effects of potassium and calcium on aerobic glycolysis in cerebral cortex slices are direct—on the pyruvate kinase—and also indirect. Calcium was shown to be inhibitive also to the activities of hexokinase, phosphoglucoisomerase, phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase and enolase of guinea pig cerebral tissue.     

The activities of fructose 1,6-diphosphatase, phosphofructokinase and phosphoenolpyruvate carboxykinase in white muscle and red muscle

1. The activities of fructose 1,6-diphosphatase were measured in extracts of muscles of various physiological function, and compared with the activities of other enzymes including phosphofructokinase, phosphoenolpyruvate carboxykinase and the lactate-dehydrogenase isoenzymes. 2. The activity of phosphofructokinase greatly exceeded that of fructose diphosphatase in all muscles tested, and it is concluded that fructose diphosphatase could not play any significant role in the regulation of fructose 6-phosphate phosphorylation in muscle. 3. Fructose-diphosphatase activity was highest in white muscle and low in red muscle. No activity was detected in heart or a deep-red skeletal muscle, rabbit semitendinosus. 4. The lactate-dehydrogenase isoenzyme ratio (activities at high and low substrate concentration) was measured in various muscles because a low ratio is characteristic of muscles that are more dependent on glycolysis for their energy production. As the ratio decreased the activity of fructose diphosphatase increased, which suggests that highest fructose-diphosphatase activity is found in muscles that depend most on glycolysis. 5. There was a good correlation between the activities of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle, where the activities of these enzymes were similar to those of liver and kidney cortex. However, the activities of pyruvate carboxylase and glucose 6-phosphatase were very low in white muscle, thereby excluding the possibility of gluconeogenesis from pyruvate and lactate. 6. It is suggested that the presence of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle may be related to operation of the alpha-glycerophosphate-dihydroxyacetone phosphate and malate-oxaloacetate cycles in this tissue.     

An investigation of lipogenic and glycolytic enzyme activity in the liver of sexually immature and mature domestic fowl

In the non-laying pullet and the cockerel it was observed that there was no significant variation in the activities of ATP citrate lyase and ;malic' enzyme whereas in the laying hen there was a significantly greater activity of both these enzymes. Parallel increases in liver lipid content in the laying hen were also observed. Three glycolytic enzymes, phosphofructokinase, fructose diphosphate aldolase and pyruvate kinase, did not exhibit any significant variation in enzyme activity with the onset of egg laying. These results are discussed in relation to the hormonal status of the birds and also the demands of egg production for lipid.     

The effects of calcium ions on the activities of trehalase, hexokinase, phosphofructokinase, fructose diphosphatase and pyruvate kinase from various muscles

1. The effects of Ca²⁺ on the activities and regulatory properties of trehalase, hexokinase, phosphofructokinase, fructose diphosphatase and pyruvate kinase from vertebrate red and white muscle and insect fibrillar and non-fibrillar muscle have been investigated. These muscles were selected because of the possible difference in the role of glycolysis in energy production in the vertebrate muscles, and the possible difference in the role of Ca²⁺ in the control of contraction in the two types of insect muscle. An increase in Ca²⁺ concentration from 0.001μm to 10μm did not modify the activities nor did it modify the regulatory properties of these enzymes from these various muscles. 2. Concentrations of Ca²⁺ above 0.1mm inhibited the activities of hexokinase and phosphofructokinase from the different muscles. It has been suggested that this inhibition may provide the basis for a theory of regulation of glycolysis (Margreth et al., 1967). If phosphofructokinase is located within the sarcoplasmic reticulum, its activity will be inhibited when the muscle is at rest, but the release of Ca²⁺ from the reticulum during contraction will lead to a stimulation of its activity and hence an increase in glycolytic flux. The distribution of hexokinase and phosphofructokinase in the various cell fractions of these muscles was very variable. In particular, both enzymes were present almost exclusively in the 100000g supernatant fraction in the extracts of insect flight muscles. Thus there is no correlation between the properties of the enzymes and their distribution in muscle. 3. It is concluded that Ca²⁺ does not control the activities of the important regulatory enzymes of glycolysis in muscle. It is suggested that in some muscles the sensitivity of the control mechanism at the level of phosphofructokinase to changes in the concentration of AMP may be increased by a process known as `substrate-cycling'.     

Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates

1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13mum produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the K(m) values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn(2+) in addition to Mg(2+) for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.     

The regulation of glycolysis in perfused locust flight muscle

Concentrations of glycolytic intermediates, amino acids and possible regulator substances were measured in extracts from locust thoracic muscles perfused under different conditions. The conversion of [(14)C]glucose into intermediates and CO(2) by muscle preparations was also followed. When muscles perfused with glucose were made anaerobic changes in metabolite concentrations occurred that could be accounted for by an activation of phosphofructokinase and pyruvate kinase. When butyrate and glucose were present in the perfusion medium the rate of glycolytic flux was lower than with glucose alone, and the aldolase reaction appeared to be inhibited. When butyrate alone was supplied to the muscle the concentrations of most glycolytic intermediates were similar to those found when glucose was supplied. Iodoacetate caused changes in concentrations of intermediates that appeared to result from inhibition of glyceraldehyde 3-phosphate dehydrogenase. Fluoroacetate-poisoned muscles showed a high citrate concentration, but no obvious site of inhibition by citrate was apparent in the glycolytic pathway. Mechanisms for control of glycolysis in locust flight muscle are discussed and related to the known properties of isolated enzymes. It is proposed that trehalase, hexokinase, phosphofructokinase, aldolase, and pyruvate kinase may be control enzymes in this tissue.     

Metabolic adaptation of the renal carbohydrate metabolism. II

Summary The effects of a high carbohydrate diet on the renal gluconeogenic and glycolytic capacities and on the activities of the main enzymes of the carbohydrate metabolism, fructose 1,6-bisphosphatase, phosphofructokinase and pyruvate kinase have been studied. These parameters have been analysed in two separate and isolated fractions of the renal tubule, the proximal convoluted (PCT) and the distal convoluted (DCT) zones. The results presented in this study show a rapid adaptation capacity of the kidney in response to the high amount of dietary carbohydrate, which are characterized by a decrease in the glucose production and fructose 1,6-bisphosphatase activity in the proximal tubules, and an increase in the glycolytic flux and phosphofructokinase and pyruvate kinase activities in the distal tubules. The changes in these enzyme activities took place only at subsaturating substrate concentrations and not at maximum velocity which suggest that they are probably due to an allosteric and/or covalent modifications and so, they are independent of variations in the cellular levels of the enzymes.     

Regulation of the skeletal muscle metabolism during hibernation of Jaculus orientalis

1. The glycolytic flow in the skeletal muscle was considerably depressed during hibernation of Jaculus orientalis. 2. Although the ATP content was not modified, the ATP/AMP ratio was twice as large than under homeothermic conditions. 3. Furthermore, the hexose phosphates were markedly depressed. 4. The total activities of the key enzymes, hexokinase, phosphofructokinase and pyruvate kinase, which are regulated through the adenylates, decreased. 5. Under in vivo conditions, taking into account the small amount of fructose-6-phosphate and the ATP/AMP ratio, it is likely that phosphofructokinase was totally inhibited, explaining the undetectable amount of fructose 1.6 bisphosphate.     

Identification of the regulatory steps in glycolysis in potato tubers

The aim of this work was to identify the regulatory reactions of glycolysis in potato tubers. The amounts of glycolytic intermediates in aerobic and anoxic tubers were measured in freeze-clamped samples of tissue. Comparison of mass—action ratios with apparent equilibrium constants showed that in vivo the reactions catalysed by glucosephosphate isomerase, phosphoglycerate mutase and enolase were close to equilibrium. The ratios fructose-1,6-bisphosphate:fructose 6-phosphate, and pyruvate:phosphoenolpyruvate, respectively, showed that the reactions catalysed by phosphofructokinase and pyruvate kinase were considerably displaced from equilibrium. Stimulation of glycolysis by placing tubers in an atmosphere of nitrogen led to significant declines in their contents of fructose-6-phosphate and phosphoenolpyruvate. It is concluded that phosphofructokinase plays a dominant role in regulating entry into glycolysis, and that pyruvate kinase may regulate exit from glycolysis and the oxidative pentose phosphate pathway. Cold-induced sweetening of the tubers is discussed in the light of the above conclusions.     

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.     

Studies of regulatory metabolism in Moniezia expansa: general considerations.

The activities of selected enzymes in the branched metabolic pathway to succinate or lactate were determined in cytosol and mitochondrial fractions. The enzymes of lowest activity in the cytosol, and thus possibly regulatory, are phosphofructokinase and pyruvate kinase. Malic enzyme activity could scarcely be detected in either compartment; phosphoenolpyruvate carboxykinase and malate dehydrogenase occur in both. The end products of metabolism are succinate and lactate; under anaerobic conditions lactate production increases whereas succinate production shows a small decrease. The presence of glucose in the medium does not influence the change, but causes an increase in total endproduct accumulation. Levels of metabolic intermediates in worms incubated aerobically and anaerobically are presented, and ‘cross-over’ plots and calculations of apparent equilibrium constants identify hexokinase, phosphofructokinase and pyruvate kinase as regulatory. Under aerobic conditions a large increase in the size of the malate pool is observed suggesting that the depression of lactate production is produced by its inhibitory effect on pyruvate kinase. Adenine nucleotide levels are maintained whether or not the worm is incubated under anaerobic conditions.     

Co-ordinate control of phosphofructokinase and pyruvate kinase by fructose diphosphate: a mechanism for amplification and step changes in the regulation of glycolysis in liver

Activation of both phosphofructokinase and pyruvate kinase by fructose diphosphate in liver provides a means of amplifying effects of other activators or inhibitors in controlling the rate of glycolysis. Two types of behavior can occur, depending on the choice of affinity constants of the two enzymes for fructose diphosphate in a simple model: (i) there may be a steady state corresponding to each value of the fructose diphosphate concentration, so that the glycolytic rate is continuously variable, or (ii) there may be two (or more) regions of stable steady states, separated by a zone of instability, so that the system shifts abruptly between low and high glycolytic rates at critical concentrations of fructose diphosphate. A low glycolytic rate corresponds to net gluconeogenesis when the gluconeogenic enzymes are included. Calculations from data from perfused liver support the proposal that the free fructose diphosphate concentration is a major factor controlling glycolysis in liver and amplifying the effect of changes in the fructose 6-phosphate concentration which occur in response to variation in the glucose concentration.     

Regulatory steps of glycolysis during environmental anoxia and muscular work in the cockle,Cardium tuberculatum: control of low and high glycolytic flux

Summary Concentrations of glycolytic intermediates, end products of anaerobic metabolism and the adenylates have been determined in the foot muscle and in the whole soft body tissue of the cockle,Cardium tuberculatum, after anoxic incubation and after the performance of vigorous escape movements. Comparison of the mass action ratios (MAR) with the equilibrium constants (Keq) showed that the reactions catalyzed by glycogen phosphorylase, hexokinase, phosphofructokinase (PFK) and pyruvate kinase (PK) were displaced from equilibrium under all physiological situations investigated.Changes in the levels of the glycolytic intermediates showed that activation of phosphofructokinase is largely responsible for the 100-fold increase of glycolytic flux in the foot muscle during exercise.Analysis of the whole soft body tissue showed that PFK is also involved in reduction of the glycolytic flux during anoxia, but a more pronounced change in the MAR occurs for PK, indicating that PK is strongly inhibited under these conditions.Differences in the regulation of glycolysis in muscular and non-muscular tissues can be related to changes in metabolite levels and to tissue-specific forms of pyruvate kinase with different regulatory properties.     

Glycosis in the eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas. Rate-limiting steps and activation at fertilization.

The content of glycolytic intermediates and of adenine nucleotides was measured in eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas, before and after fertilization. On the whole, the profile of the change in each glycolytic intermediate in Urechis eggs upon fertilization was found to be essentially similar to that in oyster eggs. Calculation of the mass action ratio for each glycolytic step from the amounts of glycolytic intermediates determined suggests that there are at least three limiting enzymes in the glycolysis system in unfertilized and fertilized eggs of each species examined. Phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11), and pyruvate kinase (EC 2.7.1.40) may be rate-limiting enzymes for the glycolysis system in Urechis eggs as well as in oyster eggs. These enzymes are thought to be activated upon fertilization, though even the reactions of the enzymes in fertilized eggs do not reach a state of equilibrium. In eggs of Urechis and oyster, phosphorylase is the first enzyme to be activated following fertilization. In Urechis eggs, pyruvate kinase is activated after the instant increase in the phosphorylase activity upon fertilization, followed by phosphofructokinase activation. In oyster eggs, however, pyruvate kinase and phosphofructokinase seem to be stimulated simultaneously, subsequent to phosphorylase activation upon fertilization. The mechanism controlling phosphorylase and pyruvate kinase activity is unknown, but the phosphofructokinase activity in both species may be regulated by the intracellular concentration of adenine nucleotides, since the enzyme activity is enhanced along with a decline in the phosphate potential in the eggs of both Urechis and of oyster.     

The inhibitory effect of various fatty acids on aerobic glycolysis in Ehrlich ascites tumour cells

Inhibition of glycolysis in Ehrlich ascites tumour cells by saturated fatty acids, added either in form of potassium salts or incorporated into phosphatidylcholine liposomes, increases with the increasing carbon atom chain length and is independent of the concentration within the range of 0.1 to 1.0 mM. In contrast, the inhibition of glycolysis in the cytosolic fraction from Ehrlich ascites cells depends on the concentration of fatty acids. The content of ATP in Ehrlich ascites cells incubated with fatty acids increases with increasing carbon atom chain length, which leads to a crossing-over in the concentrations of pyruvate and 2-phosphoenolpyruvate. Lowering of the sum of both these metabolites by palmitate and stearate points to the inhibition not only of pyruvate kinase but also of other enzymes of early steps of glycolysis. Fatty acids in intact Ehrlich ascites cells inhibit all three key glycolytic enzymes but added to the cytosolic fraction affect mainly the activity of phosphofructokinase. The inhibition of pyruvate kinase by fatty acids is smaller in the cytosolic fraction from tumour cells than from liver and muscles.     

A comparison of the properties of fructose 1,6-diphosphatase, and the activities of other key enzymes of carbohydrate metabolism, in the livers of embryonic and adult rat, sheep and domestic fowl

1. The activities of some key enzymes of glycolysis and gluconeogenesis were measured in embryonic chick, sheep and rat livers. 2. In chicken the activities of hexokinase, phosphofructokinase and pyruvate kinase are low, but those of glucose 6-phosphatase and fructose diphosphatase are very high; the converse situation exists in the rat (Burch et al. 1963), but in sheep the activities of both phosphofructokinase and fructose diphosphatase are high, and the activities of hexokinase and glucose 6-phosphatase are low. These findings are discussed in relation to carbohydrate metabolism in these embryonic livers. 3. The regulatory properties of fructose diphosphatase from the embryonic livers of these three species were compared with the properties of the enzymes from adult animals. The inhibitions by AMP and fructose diphosphate and the effects of Mg(2+) and pH on the activities of adult and foetal fructose diphosphatase are almost identical. 4. It is concluded that regulatory properties are characteristic of fructose diphosphatase from embryonic and adult tissue, and the importance of this in relation to enzyme development is discussed.     

Effects of T4 phage infection and anaerobiosis upon nucleotide pools and mutagenesis in nucleoside diphosphokinase-defective Escherichia coli strains.

Bacteriophage T4 encodes nearly all of its own enzymes for synthesizing DNA and its precursors. An exception is nucleoside diphosphokinase (ndk gene product), which catalyzes the synthesis of ribonucleoside triphosphates and deoxyribonucleoside triphosphates (dNTPs) from the corresponding diphosphates. Surprisingly, an Escherichia coli ndk deletion strain grows normally and supports T4 infection. As shown elsewhere, these ndk mutant cells display both a mutator phenotype and deoxyribonucleotide pool abnormalities. However, after T4 infection, both dNTP pools and spontaneous mutation frequencies are near normal. An E. coli strain carrying deletions in ndk and pyrA and pyrF, the structural genes for both pyruvate kinases, also grows and supports T4 infection. We examined anaerobic E. coli cultures because of reports that in anaerobiosis, pyruvate kinase represents the major route for nucleoside triphosphate synthesis in the absence of nucleoside diphosphokinase. The dNTP pool imbalances and the mutator phenotype are less pronounced in the anaerobic than in the corresponding aerobic ndk mutant strains. Anaerobic dNTP pool data, which have not been reported before, reveal a disproportionate reduction in dGTP, relative to the other pools, when aerobic and anaerobic conditions are compared. The finding that mutagenesis and pool imbalances are mitigated in both anaerobic and T4-infected cultures provides strong, if circumstantial, evidence that the mutator phenotype of ndk mutant cells is a result of the dNTP imbalance. Also, the viability of these cells indicates the existence of a second enzyme system in addition to nucleoside diphosphokinase for nucleoside triphosphate synthesis.     

Endotoxin increases the liver fructose 2,6-bisphosphate concentration in fasted rats

Following endotoxin administration to fasted rats, the liver fructose 2,6-bisphosphate level is significantly increased within 1 hr, is elevated 2.3-fold by 3 hrs, and remains elevated 2 to 3-fold for at least 24 hrs. This increase in the potent allosteric activator of phosphofructokinase occurs when there is no change in the liver Glc 6-P, glycogen or cAMP concentrations, or in the activities of phosphoenolpyruvate carboxykinase or pyruvate kinase. The increase in fructose 2,6-bisphosphate concentration accounts for the increased phosphofructokinase activity previously observed in hepatocytes isolated 18 hours following endotoxin administration to rats (1). By stimulating the phosphofructokinase/Fru 1,6-bisphosphate cycle in the direction of glycolysis, fructose 2,6-bisphosphate is likely the factor responsible for decreased gluconeogenesis in endotoxemia.     

Long-term control on renal carbohydrate metabolism--II. Effect of starve-feed cycles on renal tubule glycolysis

1. The effects of different and alternative starve-feed cycles on glycolysis from isolated renal tubules as well as the glycolytic enzymes phosphofructokinase and pyruvate kinase have been studied. Adaptive responses of renal glycolysis under the nutritional conditions mentioned are reported. 2. Renal glucose utilization increased in a linear fashion during the feeding state of the nutritional cycles, becoming twice as much in both feeding and fasting cycles. Conversely, a decrease in this metabolic pathway took place during the starve periods of the cycles. During the feed-starve cycle the decrease reached 70% in 48 hr of fasting after being fed with a high carbohydrate diet. Whereas in the opposite cycle it was almost 35%. 3. The activities of renal glycolytic enzymes, phosphofructokinase and pyruvate kinase are parallel to the glycolytic capacity of renal tubules in different nutritional conditions. These changes only occur at cellular substrate concentration. 4. The behaviour of the kinetic parameters of these enzymes throughout these experimental conditions is reported. In general, variations in Km values without changes in Vmax values take place which reflect an increase in the catalytic efficiency of the glycolytic enzymes during the feeding state and conversely a decrease during the starvation state.