Control of phosphofructokinase from rat skeletal muscle. Effects of fructose diphosphate, AMP, ATP, and citrate.

Under conditions used previously for demonstrating glycolytic oscillations in muscle extracts (pH 6.65, 0.1 to 0.5 mM ATP), phosphofructokinase from rat skeletal muscle is strongly activated by micromolar concentrations of fructose diphosphate. The activation is dependent on the presence of AMP. Activation by fructose diphosphate and AMP, and inhibition by ATP, is primarily due to large changes in the apparent affinity of the enzyme for the substrate fructose 6-phosphate. These control properties can account for the generation of glycolytic oscillations. The enzyme was also studied under conditions approximating the metabolite contents of skeletal muscle in vivo (pH 7.0, 10mM ATP, 0.1 mM fructose 6-phosphate). Under these more inhibitory conditions, phosphofructokinase is strongly activated by low concentrations of fructose diphosphate, with half-maximal activation at about 10 muM. Citrate is a potent inhibitor at physiological concentrations, whereas AMP is a strong activator. Both AMP and citrate affect the maximum velocity and have little effect on affinity of the enzyme for fructose diphosphate.     

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.     

Regulation of glycolysis and l-glycerol 3-phosphate concentration in rat epididymal adipose tissue in vitro. Role of phosphofructokinase

1. Attempts were made to define the role of phosphofructokinase in glycolytic control and the factors regulating the concentration of l-glycerol 3-phosphate in rat epididymal fat pads incubated in vitro. 2. Glycolysis rates were altered by anoxia or by additions of insulin, adrenaline or both to the incubation medium, and the changes in rate were related to changes in the steady-state concentrations of hexose phosphates, adenine nucleotides, l-glycerol 3-phosphate and citrate in the whole tissue. Measurements were also made of the lactate/pyruvate concentration ratio in the medium after incubation. 3. The mass-action ratios of phosphofructokinase, calculated from the whole-tissue concentrations of products and substrates, were less than 0.1% of the value of the ratio at pH7.4 at equilibrium. 4. Only in the presence of adrenaline could the observed stimulation of glycolytic flux be related to a possible activation of phosphofructokinase since, in this situation, the concentration of one substrate, fructose 6-phosphate, was not altered and the concentration of the other, ATP, was decreased. Increased glycolytic flux in the presence of insulin may be explained by an observed increase in the concentration of the substrate, fructose 6-phosphate. Under anaerobic conditions, glycolytic flux was decreased but this did not appear to be the result of inhibition of phosphofructokinase, since the concentrations of both substrates, fructose 6-phosphate and ATP, were decreased. The changes in glycolytic flux with insulin and anoxia may be secondary to changes in the rate of glucose uptake. 5. Changes in l-glycerol 3-phosphate concentration appear to be related both to changes in the concentration of dihydroxyacetone phosphate and to changes in the NADH/NAD(+) concentration ratio in the cytoplasm. They do not seem to be related directly to alterations in glycolytic rate.     

Interaction of D-fructose and fructose 1-phosphate with yeast phosphofructokinase and its influence on glycolytic oscillations.

Fermentation of D-fructose- and D-glucose induced glycolytic oscillations of different period lengths in Saccharomyces carlsbergensis. Recent studies suggested, that D-fructose or one of its metabolites interacted with phosphofructokinase (ATP:D-fructo-6-phosphate 1-phosphofructokinase, EC 2.7.1.11), the core of the glycolytic 'oscillator'. In order to explore the kinetics of interaction, the influence of D-fructose and fructose 1-phosphate on purified yeast phosphofructokinase was studied. D-fructose concentrations up to 0.3 mM stimulated the enzyme, while a further increase led to competitive inhibition. The Hill coefficient for fructose 6-phosphate decreased from 2.8 to 1.0. Fructose 1-phosphate acted in a similar way, up to 1 mM activation and inhibition competitive to fructose 6-phosphate at higher concentration (2.0--3.5 mM) with the same effect on the Hill coefficient. The inhibition patterns obtained with D-fructose or fructose 1-phosphate suggest a sequential random reaction mechanism of yeast phosphofructokinase with fructose 6-phosphate and MgATP2-. The mode of interaction of phosphofructokinase with D-fructose and fructose 1-phosphate is discussed. The influence of both effectors resulted in altered enzyme kinetics, which may cause the different period lengths of glycolytic oscillations.     

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.     

The activities of fructose diphosphatase in flight muscles from the bumble-bee and role of this enzyme in heat generation

1. The maximum catalytic activities of fructose diphosphatase from flight muscles of bumble-bees (Bombus spp.) are at least 30-fold those reported for the enzyme from other tissues. The maximum activity of fructose diphosphatase in the flight muscle of any particular bee is similar to that of phosphofructokinase in the same muscle, and the activity of hexokinase is similar to or greater than the activity of phosphofructokinase. There is no detectable activity of glucose 6-phosphatase and only a very low activity of glucose 6-phosphate dehydrogenase in these muscles. The activities of both fructose diphosphatase and phosphofructokinase vary inversely with the body weight of the bee, whereas that of hexokinase is relatively constant. 2. There is no significant hydrolysis of fructose 1-phosphate, fructose 6-phosphate, glucose 1,6-diphosphate and glycerol 3-phosphate by extracts of bumble-bee flight muscle. 3. Fructose 1,6-diphosphatase from bumble-bee flight muscle and from other muscles is inhibited by Mn(2+) and univalent cations; the potency of inhibition by the latter varies in the order Li(+)>Na(+)>K(+). However, the fructose diphosphatase from bumble-bee flight muscle is different from the enzyme from other tissues in that it is not inhibited by AMP. 4. The contents of ATP, hexose monophosphates, fructose diphosphate and triose phosphates in bumble-bee flight muscle showed no significant changes between rest and flight. 5. It is proposed that both fructose diphosphatase and phosphofructokinase are simultaneously active and catalyse a cycle between fructose 6-phosphate and fructose diphosphate in resting bumble-bee flight muscle. Such a cycle would produce continuous hydrolysis of ATP, with the release of energy as heat, which would help to maintain the thoracic temperature during rest periods at a level adequate for flight.     

Substrate specificity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase from potato tuber

The aim of this work was to establish the precise ionic form of the reactants used by pyrophosphate:fructose-6-phosphate phosphotransferase. The enzyme was purified to near-homogeneity from potato (Solanum tuberosum L.) tubers. Changes in enzyme activity when the pH of the assay and the concentration of fructose 6-phosphate, pyrophosphate, and magnesium are varied independently indicate that fructose 6-phosphate²⁻ and MgP₂O₇²⁻ are the reacting species in the glycolytic direction. Analogous experiments with fructose 1,6-bisphosphate, inorganic phosphate, and magnesium demonstrate that the enzyme uses fructose 1,6-bisphosphate⁴⁻, HPO₄²⁻, and Mg²⁺ in the gluconeogenic direction. The ionic species used in the glycolytic direction are comparable with those required by bacterial ATP-dependent phosphofructokinase. This is consistent with the proposal that the active site of pyrophosphate:fructose-6-phosphate phosphotransferase in plants is equivalent to that of the bacterial phosphofructokinase (SM Carlisle et al. [1990] J Biol Chem 265: 18366-18371).     

Biochemical mechanisms of red blood cell 2,3-diphosphoglycerate increase at high altitude

Red blood cell 2,3 diphosphoglycerate (2,3-DPG) levels increase after ascent to high altitude. Studies were undertaken to identify the biochemical mechanisms responsible for eliciting the 2,3-DPG response in several types of subjects. These included (1) short-term exposure to 3400 m in ten subjects; (2) exposure to 4300 m in an additional ten subjects; (3) studies in 28 high-altitude normal residents of 3100 m; and (4) studies in 28 high-altitude residents with chronic mountain polycythemia. Controls were 41 residents of 240 m. Regression analysis identified the glycolytic variables, termed “key variables,” on which variation in 2,3-DPG levels was dependent (P < .05). Key variables common to the short-term studies were glucose-6-phosphate, phosphoenolpyruvate, and the ratio of the levels of adenosine diphosphate to adenosine triphosphate. The positions of these key variables in the glycolytic pathway and their mean levels suggest erythrocyte hexokinase and pyruvate kinase activation as possible enzymatic mechanisms. Key variables unique to the 3400 m study suggested phosphofructokinase activation also acted to increase 2,3-DPG levels. 2,3-DPG levels in the normal 3100 m residents were not different from low-altitude values, and 2,3-DPG levels in these samples did not appear to be dependent on any of the glycolytic variables examined. Among the high-altitude residents with polycythemia, higher 2,3-DPG levels were dependent on glucose-6-phosphate, fructose diphosphate, dihydroxyacetone phosphate, and the ratio of adenosine diphosphate to adenosine triphosphate levels. The positions of these variables in the glycolytic pathway and their mean levels suggested activation of the hexokinase and phosphofructokinase enzymes.     

Regulation of climacteric respiration in ripening avocado fruit

Ripening of avocado fruit is associated with a dramatic increase in respiration. In vivo³¹P nuclear magnetic resonance spectroscopy revealed large increases in ATP levels accompanying the increase in respiration. Both glycolytic enzymes, phosphofructokinase, and pyrophosphate: fructose-6-phosphate phosphotransferase were present in avocado fruit with the latter activity being highly stimulated by fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate levels increased approximately 90% at the onset of ripening, suggesting that the respiratory increase in ripening avocado fruit may be regulated by the activation of pyrophosphate:fructose-6-phosphate phosphotransferase by an increase in fructose 2,6-bisphosphate.     

Affinity chromatography of phosphofructokinase.

The behavior of mammalian phosphofructokinase on immobilized adenine nucleotides was investigated. Three different insolubilized ligands were compared using a pure rabbit muscle phosphofructokinase. N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose bound at least 90 times more enzyme than either N⁶-(6-aminohexyl)-AMP-agarose or ATP-adipic acid hydrazide-Sepharose. The elution of phosphofructokinase from the ATP-Sepharose with various metabolites and combinations of metabolites was investigated. The enzyme is eluted specifically from N⁶-[(6-aminohexyl)-carbamoyl]-ATP-Sepharose with a mixture of 25 μm each of fructose 6-phosphate and ADP (±Mg²⁺). The enzyme is not eluted either with ATP (25 μm), fructose 1,6-diphosphate (1 mm), ADP (25 μm), fructose 6-phosphate (1 mm) alone, or with a mixture of fructose 1,6-diphosphate (25 μm) and ATP (25 μm). The recovery of bound enzyme was usually greater than 90%. A mixture of glucose 6-phosphate and ADP or a mixture of IDP and fructose 6-phosphate also elutes the enzyme, but the recovery with these eluants was only about 40%. It was concluded that the “dead-end” complex is the most effective in the elution. Using this method, phosphofructokinase has been prepared in an essentially homogeneous form from muscle and brain of rabbit and rat. The overall isolation procedure involves a high speed centrifugation of crude extracts which sediments phosphofructokinase as a pellet, followed with adsorption on N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose and specific elution with the mixture of fructose 6-phosphate and ADP.     

Oscillations of the glycolytic pathway and the purine nucleotide cycle.

In a treatment modeled after the oscillatory behavior of the glycolytic pathway and the purine nucleotide cycle observed in skeletal muscle extracts, it is shown that the basis of the oscillations is the AMP-dependent activation of phosphofructokinase by fructose diphosphate. Control of phosphofructokinase by the adenine nucleotides alone leads to the establishment of a steady state. Whether steady state or oscillatory behavior occurs depends in part on the activity of glyceraldehyde-3-phosphate dehydrogenase, which controls the rate of removal of fructose diphosphate. Under appropriate conditions oscillatory behavior can maintain a higher [ATP]/[ADP] ratio than steady state behavior. Viewed in the context of conditions that may be encountered in skeletal muscle in vivo, oscillatory behavior of glycolysis is shown to have additional advantages for maintaining a high [ATP]/[ADP] ratio.     

Metabolic Regulation in the Senescing Tobacco Leaf: II. Changes in Glycolytic Metabolite Levels in the Detached Leaf

Yellowing of detached mature tobacco leaves standing in water in the dark was accompanied by a strong “climacteric rise” in respiration rate. During this period the ATP level and energy charge of the adenylate system also rose. The levels of glycolytic intermediates between glucose 1-phosphate and triose phosphates rose, those between 3-phosphoglycerate and phosphoenolpyruvate fell, and pyruvate rose. On the assumption of a drop in NAD/NADH ratio, as found by other workers in wheat leaves, the reverse crossover between triose phosphates and 3-phospholglycerate was attributed to inhibition of glyceraldehyde 3-phosphate dehydrogenase. The forward crossover between phosphoenolpyruvate and pyruvate was taken to indicate activation of pyruvate kinase, possibly by fructose diphosphate. Secondary large rises in pyruvate and fructose diphosphate occurred well after the climacteric peak had been passed. No evidence was found for participation of phosphofructokinase in metabolic control in the yellowing leaf. Possible limitations to the use of the crossover theorem in the present situation, such as changes in compartmentation and in flux through branch points, are emphasized.     

Phosphofructokinase from bumblebee flight muscle. Molecular and catalytic properties and role of the enzyme in regulation of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle

Phosphofructokinase from the flight muscle of bumblebee was purified to homogeneity and its molecular and catalytic properties are presented. The kinetic behavior studies at pH 8.0 are consistent with random or compulsory-order ternary complex. At pH 7.4 the enzyme displays regulatory behavior with respect to both substrates, cooperativity toward fructose 6-phosphate, and inhibition by high concentration of ATP. Determinations of glycolytic intermediates in the flight muscle of insects exposed to low and normal temperatures showed statistically significant increases in the concentrations of AMP, fructose 2,6-bisphosphate, and glucose 6-phosphate during flight at 25 degrees C or rest at 5 degrees C. Measuring the activity of phosphofructokinase and fructose 1,6-bisphosphatase at 25 and 7.5 degrees C, in the presence of physiological concentrations of substrates and key effectors found in the muscle of bumblebee kept under different environmental temperatures and activity levels, suggests that the temperature dependence of fructose 6-phosphate/fructose 1,6-bisphosphate cycling may be regulated by fluctuation of fructose 2,6-bisphosphate concentration and changes in the affinity of both enzymes for substrates and effectors. Moreover, in the presence of in vivo concentrations of substrates, phosphofructokinase is inactive in the absence of fructose 2,6-bisphosphate.     

Control of glycolysis and gluconeogenesis in rat kidney cortex slices

1. Glucose uptake or glucose formation has been studied in kidney cortex slices to investigate metabolic control of phosphofructokinase and fructose-diphosphatase activities. 2. Glucose uptake is increased and glucose formation is decreased by anoxia, cyanide or an uncoupling agent. Under these conditions the intracellular concentrations of glucose 6-phosphate and ATP decreased whereas that of fructose diphosphate either increased or remained constant, and the concentrations of AMP and ADP increased. 3. Glucose uptake was decreased, and glucose formation from glycerol or dihydroxyacetone was increased, by the presence of ketone bodies or fatty acids, or after starvation of the donor animal. Under these conditions, the concentrations of glucose 6-phosphate and citrate were increased, whereas those of fructose diphosphate and the adenine nucleotides were unchanged (see also Newsholme & Underwood, 1966). 4. It is concluded that anoxia and cell poisons increase glucose uptake and decrease gluconeogenesis by stimulating phosphofructokinase and inhibiting fructose diphosphatase, whereas ketone bodies, fatty acids or starvation increase gluconeogenesis and decrease glucose uptake through the citrate inhibition of phosphofructokinase.     

The effect of pretreatment with pentobarbital on the extent of [14C] incorporation from [U-14C]glucose into various rat brain glycolytic intermediates: relevance to regulation at hexokinase and phosphofrucktokinase

In the present investigation we monitored the incorporation of [¹⁴C] from [U-¹⁴C]glucose into various rat brain glycolytic intermediates of conscious and pentobarbital-anesthetized animals. Labeled glucose was delivered to brain by single bolus intracarotid injection and brain tissue was subsequently prepared at 15, 30 and 45 sec by freeze-blowing. Glycolytic intermediates were then separated by column chromatography. Our results showed a gradual decrease with time of¹⁴C-labeled glucose which gave a calculated rate for glucose metabolism of 0.86 mol/min/g and 0.56 mol/min/g in conscious and anesthetized animals, respectively. Compared to the results obtained using conscious animals the administration of pentobarbital not only resulted in a significant attenuation of the rate of glucose metabolism but also caused a similar reduction in the amount of¹⁴C incorporated into several glycolytic intermediates. These intermediates included: glucose 6-phosphate, fructose 6-phosphate, fructose, 1,6 diphosphate, dihydroxyacetone phosphate and post glycolytic compounds. In addition, pretreatment with pentobarbital resulted in a 75% increase in the endogenous concentration of glucose, 10% increase in glucose 6-phosphate, no change in fructose 6-phosphate and 42% decrease in lactate compared to levels in brains obtained from conscious animals. These results are discussed in relation to control of glycolysis through coupled regulation at hexokinase-phosphofructokinase.     

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.     

Ribose 1,5-bisphosphate regulates rat kidney cortex phosphofructokinase

Phosphofructokinase (EC 2.7.1.11) is a major enzyme of the glycolytic pathway, catalyzing the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate. In this study, we demonstrated the effect of ribose 1,5-bisphosphate on phosphofructokinase purified from rat kidney cortex. Ribose 1,5-bisphosphate relieved the phosphofructokinase from ATP inhibition and increased the affinity for fructose 6-phosphate at nanomolar concentrations. These activating effects of ribose 1,5-bisphosphate were enhanced in the presence of AMP. Ribose 1,5-bisphosphate reduced the inhibition of the phosphofructokinase induced by citrate. These results suggest that ribose 1,5-bisphosphate is an activator of rat kidney cortex phosphofructokinase and synergistically regulates the enzyme activity with AMP.     

Metabolic profile of wound-induced changes in primary carbon metabolism in sugarbeet root

Injury to plant products by harvest and postharvest operations induces respiration rate and increases the demand for respiratory substrates. Alterations in primary carbon metabolism are likely to support the elevated demand for respiratory substrates, although the nature of these alterations is unknown. To gain insight into the metabolic changes that occur to provide substrates for wound-induced increases in respiration, changes in the concentrations of compounds that are substrates, intermediates or cofactors in the respiratory pathway were determined in sugarbeet (Beta vulgaris L.) roots in the 4 days following injury. Both wounded and unwounded tissues of wounded roots were analyzed to provide information about localized and systemic changes that occur after wounding. In wounded tissue, respiration increased an average of 186%, fructose, glucose 6-phosphate, ADP and UDP concentrations increased, and fructose 1,6-bisphosphate, triose phosphate, citrate, isocitrate, succinate, ATP, UTP and NAD⁺ concentrations decreased. In the non-wounded tissue of wounded roots, respiration rate increased an average of 21%, glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and ADP concentrations increased, and isocitrate, UTP, NAD⁺, NADP⁺, and NADPH concentrations declined. Changes in respiration rate and metabolite concentrations indicated that localized and systemic changes in primary carbon metabolism occurred in response to injury. In wounded tissue, metabolite concentration changes suggested that activities of the early glycolytic enzymes, fructokinase, phosphofructokinase, phosphoglucose isomerase, and phosphoglucomutase were limiting carbon flow through glycolysis. These restrictions in the respiratory pathway, however, were likely overcome by use of metabolic bypasses that allowed carbon compounds to enter the pathway at glycolytic and tricarboxylic acid (TCA) cycle downstream locations. In non-wounded tissue of wounded roots, metabolic concentration changes suggested that glycolysis and the TCA cycle were generally capable of supporting the small systemic elevation in respiration rate. Although the mechanism by which respiration is regulated in wounded sugarbeet roots is unknown, localized and systemic elevations in respiration were positively associated with one or more indicators of cellular redox status.     

A model study of the fructose diphosphatase-phosphofructokinase substrate cycle

A fructose diphosphatase–phosphofructokinase substrate cycle has been reconstructed in vitro to provide a system that recycles fructose 6-phosphate and hydrolyses ATP to ADP and P_i. The concerted actions of glucose phosphate isomerase, phosphofructokinase, aldolase and triose phosphate isomerase catalysed the loss of ³H from [5-³H,U-¹⁴C]glucose 6-phosphate. This was used as the basis of a method for the estimation of the fructose diphosphatase–phosphofructokinase substrate cycle. For the reconstructed cycle, the rate of decrease of the ³H/¹⁴C ratio in [5-³H,U-¹⁴C]hexose 6-phosphate was proportional to the rate of fructose 6-phosphate substrate cycling. A detailed theoretical treatment of this relationship is developed, which enables the rate of substrate cycling to be determined in vivo.     

Control of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in isolated hepatocytes by glucose and glucagon. Role of a low-molecular-weight stimulator of phosphofructokinase

1. Recycling of metabolites between fructose 6-phosphate and triose phosphates has been investigated in isolated hepatocytes by the randomization of carbon between C₍₁₎ and C₍₆₎ of glucose formed from [1-¹⁴C]galactose. 2. Randomization of carbon atoms was regularly observed with hepatocytes isolated from fed rats and was then little influenced by the concentration of glucose in the incubation medium. It was decreased by about 50% in the presence of glucagon. 3. Randomization of carbon atoms by hepatocytes isolated from starved rats was barely detectable at physiological concentrations of glucose in the incubation medium, but was greatly increased with increasing glucose concentrations. It was nearly completely suppressed by glucagon. These large changes can be attributed to parallel variations in the activity of phosphofructokinase. 4. The main factors that appear to control the activity of phosphofructokinase under these experimental conditions are the concentration of fructose 6-phosphate, the concentration of fructose 1,6-bisphosphate and also the affinity of the enzyme for fructose 6-phosphate. 5. The affinity of phosphofructokinase for fructose 6-phosphate was diminished by incubation of the cells in the presence of glucagon and also by filtration of an extract of hepatocytes through Sephadex G-25 and by purification of the enzyme. When assayed at 0.25 or 0.5mm-fructose 6-phosphate, the activity of phosphofructokinase present in a liver Sephadex filtrate was increased by a low-molecular-weight effector, which could be isolated from a liver extract by ultrafiltration, gel filtration or heat treatment, but was rapidly destroyed in trichloroacetic acid, even in the cold. This effector appears to be a highly acid-labile phosphoric ester. Its concentration was greatly increased in hepatocytes incubated in the presence of glucose and was decreased in the presence of glucagon.