2,3-Bisphosphoglycerate,fructose, 2,6-bisphosphate and glucose 1,6-bisphosphate during maturation of reticulocytes with low 2,3-bisphosphoglycerate content

In contrast to the species with erythrocytes of high 2,3-bisphosphoglycerate content, in the sheep the concentration of 2,3-bisphosphoglycerate decreases during maturation of reticulocytes. The decrease can be explained by the drop of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios that result from the decline of phosphofructokinase, pyruvate kinase, phosphoglycerate mutase and the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase. The concentrations of fructose 2,6-bisphosphate and aldohexose 1,6-bisphosphates also decrease during sheep reticulocyte maturation in parallel to the 6-phosphofructo 2-kinase and the glucose 1,6-bisphosphate synthase activities.     

Regulation by glucagon of hepatic pyruvate kinase, 6-phosphofructo 1-kinase, and fructose-1,6-bisphosphatase

Glucagon stimulates gluconeogenesis in part by decreasing the rate of phosphoenolpyruvate disposal by pyruvate kinase. Glucagon, via cyclic AMP (cAMP) and the cAMP-dependent protein kinase, enhances phosphorylation of pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase. Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased recycling of phosphoenolpyruvate to pyruvate and enhanced glucose synthesis. Although phosphorylation of 6-phosphofructo 1-kinase and fructose-1,6-bisphosphatase is catalyzed in vitro by the cAMP-dependent protein kinase, the role of phosphorylation in regulating the activity of and flux through these enzymes in intact cells is uncertain. Glucagon regulation of these two enzyme activities is brought about primarily by changes in the level of a novel sugar diphosphate, fructose 2,6-bisphosphate. This compound is an activator of phosphofructokinase and an inhibitor of fructose-1,6-bisphosphatase; it also potentiates the effect of AMP on both enzymes. Glucagon addition to isolated liver systems results in a greater than 90% decrease in the level of this compound. This effect explains in large part the effect of glucagon to enhance flux through fructose-1,6-bisphosphatase and to suppress flux through phosphofructokinase. The discovery of fructose 2,6-bisphosphate has greatly furthered our understanding of regulation at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle.     

A binding study of the interaction of beta-D-fructose 2,6-bisphosphate with phosphofructokinase and fructose-1,6-bisphosphatase

The binding of beta-D-fructose 2,6-bisphosphate to rabbit muscle phosphofructokinase and rabbit liver fructose-1,6-bisphosphatase was studied using the column centrifugation procedure (Penefsky, H. S., (1977) J. Biol. Chem. 252, 2891-2899). Phosphofructokinase binds 1 mol of fructose 2,6-bisphosphate/mol of protomer (Mr = 80,000). The Scatchard plots of the binding of fructose 2,6-bisphosphate to phosphofructokinase are nonlinear in the presence of three different buffer systems and appear to exhibit negative cooperativity. Fructose 1,6-bisphosphate and glucose 1,6-bisphosphate inhibit the binding of fructose-2,6-P2 with Ki values of 15 and 280 microM, respectively. Sedoheptulose 1,7-bisphosphate, ATP, and high concentrations of phosphate also inhibit the binding. Other metabolites including fructose-6-P, AMP, and citrate show little effect. Fructose-1,6-bisphosphatase binds 1 mol of fructose 2,6-bisphosphate/mol of subunit (Mr = 35,000) with an affinity constant of 1.5 X 10(6) M-1. Fructose 1,6-bisphosphate, fructose-6-P, and phosphate are competitive inhibitors with Ki values of 4, 2.7, and 230 microM, respectively. Sedoheptulose 1,7-bisphosphate (1 mM) inhibits approximately 50% of the binding of fructose 1,6-bisphosphate to fructose bisphosphatase, but AMP has no effect. Mn2+, Co2+, and a high concentration of Mg2+ inhibit the binding. Thus, we may conclude that fructose 2,6-bisphosphate binds to phosphofructokinase at the same allosteric site for fructose 1,6-bisphosphate while it binds to the catalytic site of fructose-1,6-bisphosphatase.     

Induction of pyrophosphate:fructose 6-phosphate 1-phosphotransferase by anoxia in rice seedlings

Rice (Oryza sativa) seeds were imbibed for 3 days and the seedlings were further incubated for 8 days in the presence of either air or nitrogen. In aerobiosis, the specific activity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase and that of the ATP-dependent phosphofructokinase increased about fourfold. In anaerobiosis, the specific activity of ATP-dependent phosphofructokinase remained stable, whereas that of pyrophosphate:fructose 6-phosphate 1-phosphotransferase increased as much as in the presence of oxygen and there was also a fourfold increase in the concentration of fructose 2,6-bisphosphate, a potent stimulator of that enzyme. These data suggest a preferential involvement of pyrophosphate:fructose 6-phosphate 1-phosphotransferase rather than of ATP-dependent phosphofructokinase in glycolysis during anaerobiosis.     

Regulation of adult and fetal myocardial phosphofructokinase. Relief of cooperativity and competition between fructose 2,6-bisphosphate, ATP, and citrate

To clarify the physiological role of fructose 2,6-bisphosphate in the perinatal switching of myocardial fuels from carbohydrate to fatty acids, the kinetic effects of fructose 2,6-bisphosphate on phosphofructokinase purified from fetal and adult rat hearts were compared. For both enzymes at physiological pH and ATP concentrations, 1 microM fructose 2,6-bisphosphate induced a greater than 10-fold reduction in S0.5 for fructose 6-phosphate and it completely eliminated subunit cooperativity. Fructose 2,6-bisphosphate may thereby reduce the influence of changes in fructose 6-phosphate concentration on phosphofructokinase activity. Based on double-reciprocal plots and ATP inhibition studies, adult heart phosphofructokinase activity is more sensitive to physiological changes in ATP and citrate concentrations than to changes in fructose 2,6-bisphosphate concentrations. Fetal heart phosphofructokinase is less sensitive to ATP concentration above 5 mM and equally sensitive to citrate inhibition. The fetal enzyme has up to a 15-fold lower affinity for fructose 2,6-bisphosphate, rendering it more sensitive to changes in fructose 2,6-bisphosphate concentration than adult heart phosphofructokinase. Together, these factors allow greater phosphofructokinase activity in fetal heart while retaining sensitive metabolic control. In both fetal and adult heart, fructose 2,6-bisphosphate is primarily permissive: it abolishes subunit cooperativity and in its presence phosphofructokinase activity is extraordinarily sensitive to both the energy balance of the cell as reflected in ATP concentration and the availability of other fuels as reflected in cytosolic citrate concentration.     

Inhibition of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

Rat liver fructose-1,6-bisphosphatase, which was assayed by measuring the release of 32P from fructose 1,6-[1-32P]bisphosphate at pH 7.5, exhibited hyperbolic kinetics with regard to its substrate. beta-D-Fructose 2,6-bisphosphate, an activator of hepatic phosphofructokinase, was found to be a potent inhibitor of the enzyme. The inhibition was competitive in nature and the Ki was estimated to be 0.5 microM. The Hill coefficient for the reaction was 1.0 in the presence and absence of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate also enhanced inhibition of the enzyme by the allosteric inhibitor AMP. The possible role of fructose 2,6-bisphosphate in the regulation of substrate cycling at the fructose-1,6-bisphosphatase step is discussed.     

Fructose 2,6-bisphosphate. A new activator of phosphofructokinase

A new activator of rat liver phosphofructokinase was partially purified from rat hepatocyte extracts by DEAE-Sephadex chromatography. The activator, which eluted in the sugar diphosphate region, was sensitive to acid treatment but resistant to heating in alkali. Mild acid hydrolysis resulted in the appearance of a sugar monophosphate which was identified as fructose 6-phosphate by gas chromatography/mass spectroscopy. These observations suggest that the activator is fructose 2,6-bisphosphate. This compound was synthesized by first reacting fructose 1,6-bisphosphate with dicyclohexylcarbodiimide and then treating the cyclic intermediate with alkali. The structure of the synthetic compound was definitively identified as fructose 2,6-bisphosphate by 13C NMR spectroscopy. Fructose 2,6-bisphosphate had properties identical with those of the activator purified from hepatocyte extracts. It activated both the rat liver and rabbit skeletal muscle enzyme in the 0.1 microM range and was several orders of magnitude more effective than fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate was not a substrate for aldolase or fructose 1,6-bisphosphatase. It is likely that this new activator is an important physiologic factor of phosphofructokinase in vivo.     

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.     

The characteristics of pyrophosphate: D-fructose-6-phosphate 1-phosphotransferases from Sansevieria trifasciata leaves and Phaseolus coccineus stems

Three different molecular forms of pyrophosphate-dependent phosphofructokinase have been isolated: one from Sansevieria trifasciata leaves and two from Phaseolus coccineus stems. The form isolated from S. trifasciata has the molecular weight of about 115,000. The apparent molecular weights for the two forms from mung bean were approximately 220,000 and 450,000. All three forms have the same pH optima, an absolute requirement for Mg2+ ions both in the forward and reverse reaction, but differ in their sensitivity toward fructose 2,6-bisphosphate. Kinetic properties of the partially purified enzymes have been investigated in the presence and absence of fructose 2,6-bisphosphate. Pyrophosphate-dependent phosphofructokinase from S. trifasciata exhibited hyperbolic kinetics with all substrates tested. The saturation curves of the enzyme (form A) from mung bean for pyrophosphate, fructose 6-phosphate and fructose 1,6-bisphosphate were sigmoidal in the absence of fructose 2,6-bisphosphate. In the presence of fructose 2,6-bisphosphate these kinetics became hyperbolic.     

Fructose 2,6-bisphosphate, sugar phosphates and adenine nucleotides in the regulation of glucose metabolism in the lactating rat mammary gland

Fructose 2,6-bisphosphate is present in the rat mammary gland, rising from a value of 1.4 nmol/g in pregnancy to 4.3 nmol/g tissue at 14 days lactation; the equivalent values calculated/ml intracellular water are 5.2 and 11.6 nmol, respectively. The tissue content of fructose 6-phosphate, fructose 1,6-bisphosphate, ATP and phosphoenolpyruvate remain relatively constant in the transition from pregnancy to the height of lactation. The changes in AMP, cyclic AMP, and citrate content of the mammary gland during lactation are such as to promote an increase in fructose 2,6-bisphosphate formation and flux through phosphofructokinase.     

A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Application to a microassay of fructose 2,6-bisphosphate

Pyrophosphate : fructose-6-phosphate phosphotransferase (PPi-PFK) has been purified 150-fold from potato tubers and the kinetic properties of the purified enzyme have been investigated both in the forward and the reverse direction. Saturation curves for fructose 6-phosphate and also for fructose 1,6-bisphosphate were sigmoidal whereas those for PPi and Pi were hyperbolic. In the presence of fructose 2,6-bisphosphate, the affinity for fructose 6-phosphate and for fructose 1,6-bisphosphate were greatly increased and the kinetics became Micha?lian. The effect of fructose 2,6-bisphosphate was increased by the presence of fructose 6-phosphate and decreased by the presence of Pi. Consequently, the Ka for fructose 2,6-bisphosphate was as low as 5 nM for the forward reaction and reached 150 nM for the reverse reaction. On the basis of these properties, a procedure allowing one to measure fructose 2,6-bisphosphate in amounts lower than a picomole, is described.     

Study on the initial kinetics of yeast phosphofructokinase by stopped-flow measurements

The initial kinetics of yeast phosphofructokinase was studied by stopped-flow measurements over an enzyme concentration range from 0.5 mg/ml to 0.01 mg/ml. Before attaining the steady state the reaction showed a lag phase in the product formation, the duration of which was found to decrease with increasing enzyme concentration. The lag phase disappeared after preincubation of the enzyme for at least five minutes with either fructose 6-phosphate, fructose 1,6-bisphosphate or fructose 2,6-bisphosphate. Preincubation of the enzyme with either AMP or ADP resulted in a reduction of this phase, while ATP was without effect. Simultaneous addition of fructose 1,6-bisphosphate to the reaction mixture of the enzyme causes a significant shortening of the transient phase, whereas micromolar concentrations of fructose 2,6-bisphosphate are capable of abolishing the lag phase completely. The occurrence of an initial transient phase suggests that the enzyme after starting the reaction converts from a state of low activity to one of high activity. This conversion mainly depends on the concentration of fructose 1,6-bisphosphate generated in the course of the reaction. In addition an association reaction of the enzyme seems to be involved in the process of conversion of the phosphofructokinase during the initial transient phase.     

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.     

Kinetic properties of pyrophosphate:fructose-6-phosphate phosphotransferase from germinating castor bean endosperm

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was purified over 500-cold from endosperm of germinating castor bean (Ricinus commiunis L. var. Hale). The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3 to 7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate, and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nanomolar. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e. fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e. fructose 1,6-bisphosphate production. The possible roles of fructose 2,6-bisphosphate, fructose 6-phosphate, and phosphate in the control of PFP are discussed.     

Age-linked alterations in fructose-2,6-bisphosphate-induced modulation of rat muscle phosphofructokinase

Native muscle phosphofructokinase (PFK: EC 2.7.1.11) isolated from 25- and 100-week-old rats was subjected to in vitro studies on fructose-2,6-bisphosphate-induced alterations in the regulatory roles of other key metabolic modulators of this enzyme. Although fructose 2,6-bisphosphate-mediated reversal of citrate inhibition did not show any age-related difference, synergism with glucose-1,6-bisphosphate effect was found to be slightly increased with the enzyme of 100-week-old rats. In addition, apart from a significant decrease in the extent of fructose-2,6-bisphosphate activation, synergism with AMP activation and reversal of ATP and pyridoxal-5-phosphate inhibitions were observed to be decreased markedly with the enzyme of 100-week-old rats in comparison with that of 25-week-old rats. Such age-dependent alterations in muscle PFK provide evidence for conformational modification in this enzyme as a function of age.     

Effects of various metabolic conditions and of the trivalent arsenical melarsen oxide on the intracellular levels of fructose 2,6-bisphosphate and of glycolytic intermediates in Trypanosoma brucei

Upon differential centrifugation of cell-free extracts of Trypanosoma brucei, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase behaved as cytosolic enzymes. The two activities could be separated from each other by chromatography on both blue Sepharose and anion exchangers. 6-phosphofructo-2-kinase had a Km for both its substrates in the millimolar range. Its activity was dependent on the presence of inorganic phosphate and was inhibited by phosphoenolpyruvate but not by citrate or glycerol 3-phosphate. The Km of fructose-2,6-bisphosphatase was 7 microM; this enzyme was inhibited by fructose 1,6-bisphosphate (Ki = 10 microM) and, less potently, by fructose 6-phosphate, phosphoenolpyruvate and glycerol 3-phosphate. Melarsen oxide inhibited 6-phosphofructo-2-kinase (Ki less than 1 microM) and fructose-2,6-bisphosphatase (Ki = 2 microM) much more potently than pyruvate kinase (Ki greater than 100 microM). The intracellular concentrations of fructose 2,6-bisphosphate and hexose 6-phosphate were highest with glucose, intermediate with fructose and lowest with glycerol and dihydroxyacetone as glycolytic substrates. When added with glucose, salicylhydroxamic acid caused a decrease in the concentration of fructose 2,6-bisphosphate, ATP, hexose 6-phosphate and fructose 1,6-bisphosphate. These studies indicate that the concentration of fructose 2,6-bisphosphate is mainly controlled by the concentration of the substrates of 6-phosphofructo-2-kinase. The changes in the concentration of phosphoenolpyruvate were in agreement with the stimulatory effect of fructose 2,6-bisphosphate on pyruvate kinase. At micromolar concentrations, melarsen oxide blocked almost completely the formation of fructose 2,6-bisphosphate induced by glucose, without changing the intracellular concentrations of ATP and of hexose 6-phosphates. At higher concentrations (3-10 microM), this drug caused cell lysis, a proportional decrease in the glycolytic flux, as well as an increase in the phosphoenolypyruvate concentrations which was restricted to the extracellular compartment. Similar changes were induced by digitonin. It is concluded that the lytic effect of melarsen oxide on the bloodstream form of T. brucei is not the result of an inhibition of pyruvate kinase.     

Kinetic studies on the activation of pyrophosphate-dependent phosphofructokinase from mung bean by fructose 2,6-bisphosphate and related compounds

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was purified from the mung bean Phaseolus aureus. The enzyme is activated by fructose 2,6-bisphosphate at nanomolar concentrations. The enzyme exhibits Michaelis-Menten kinetics, and the reaction mechanism, deduced from initial velocity studies in the absence of inhibitors as well as product and dead-end inhibition studies, is rapid equilibrium random in the presence and absence of fructose 2,6-bisphosphate. In the direction of fructose 6-phosphate phosphorylation, saturating fructose 2,6-bisphosphate (1 microM) increases V congruent to 9-fold and increases V/KMgPPi and V/KF6P about 30-fold. In the reverse direction (phosphate phosphorylation), the same concentration of activator has little if any effect on V or the Km for inorganic phosphate (Pi) and Mg2+ but does increase V/KFBP about 42-fold. No changes were observed in any of the other rate constants. The binding affinity of fructose 2,6-bisphosphate to all enzyme forms is identical. The activator site of the mung bean PPi-PFK binds fructose 2,6-bisphosphate with a Kact of 30 nM with the 2,5-anhydro-D-glucitol 1,6-bisphosphate (the most effective analogue) 33-fold less tightly. Of the alkanediol bisphosphate series, 1,4-butanediol bisphosphate exhibited the tightest binding (Kact congruent to 3 microM). These and a series of other activating analogues are discussed in relation to the activator site.     

Interaction of fructose 2,6-bisphosphate and AMP with fructose-1,6-bisphosphatase as studied by nuclear magnetic resonance spectroscopy

The interaction of AMP and fructose 2,6-bisphosphate with rabbit liver fructose-1,6-bisphosphatase has been investigated by proton nuclear magnetic resonance spectroscopy (1H NMR). The temperature dependence of the line widths of the proton resonances of AMP as a function of fructose-1,6-bisphosphatase concentration indicates that the nucleotide C2 proton is in fast exchange on the NMR time scale while the C8 proton is exchange limit. The exchange rate constant, koff, has been calculated for the adenine C8 proton and is 1900 s-1. Binding of fructose 6-phosphate and inorganic phosphate, or the regulatory inhibitor, fructose 2,6-bisphosphate, results in a decrease in the dissociation rate constant for AMP from fructose-1,6-bisphosphatase, as indicated by the sharpened AMP signals. A temperature dependence experiment indicates that the AMP protons are in slow exchange when AMP dissociates from the ternary complex. The rate constant for dissociation of AMP from the enzyme.AMP.fructose 2,6-bisphosphate complex is 70 s-1, 27-fold lower than that of AMP from the binary complex. These results are sufficient to explain the enhanced binding of AMP in the presence of fructose 2,6-bisphosphate and, therefore, the synergistic inhibition of fructose-1,6-bisphosphatase observed with these two regulatory ligands. Binding of fructose 2,6-bisphosphate to the enzyme results in broadening of the ligand proton signals. The effect of AMP on the binding of fructose 2,6-bisphosphate to the enzyme has also been investigated. An additional line width broadening of all the fructose 2,6-bisphosphate protons has been observed in the presence of AMP. The assignment of these signals to the sugar was accomplished by two-dimensional proton-proton correlated spectra (two-dimensional COSY) NMR. From these data, it is concluded that AMP can also affect fructose 2,6-bisphosphate binding to fructose-1,6-bisphosphatase.     

Effects of fructose 1,6-bisphosphate on the activation of yeast phosphofructokinase by fructose 2,6-bisphosphate and AMP

Fructose 1,6-bisphosphate decreases the activation of yeast 6-phosphofructokinase (ATP:fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) by fructose 2,6-bisphosphate, especially at cellular substrate concentrations. AMP activation of the enzyme is not influenced by fructose 1,6-bisphosphate. Inorganic phosphate increases the activation by fructose 2,6-bisphosphate and augments the deactivation of the fructose 2,6-bisphosphate activated enzyme by fructose 1,6-bisphosphate. Because various states of yeast glucose metabolism differ in the levels of the two fructose bisphosphates, the observed interactions might be of regulatory significance.     

Phosphofructokinase in rat lung during perinatal development: characterization of subunit composition and regulation by fructose 2,6-bisphosphate and glucose 1,6-bisphosphate

The subunit composition of phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11) was studied in rat lung during perinatal development. No change in subunit composition during this period was observed. The three subunits of phosphofructokinase (L, M and C) were present in a ratio of approx. 65:25:10, respectively. In addition the levels of two effectors of phosphofructokinase were determined in rat lung during perinatal development: glucose 1,6-bisphosphate and fructose 2,6-bisphosphate. Until day 20 of gestation (term is 22 days) the glucose 1,6-bisphosphate level remains relatively constant (approx. 0.55 mumol/g protein), decreases before birth and increases sharply up to 1.04 mumol/g protein 2 days after birth. The amount of fructose 2,6-bisphosphate in rat lung shows a different developmental profile. A small peak is shown at day 17 of gestation whereas a larger peak up to 36.4 nmol/g protein is shown at days 20 and 21 of gestation. The time of maximal fructose 2,6-bisphosphate content corresponds with the time of glycogen breakdown and acceleration of surfactant synthesis in prenatal rat lung. Both glucose 1,6-bisphosphate and fructose 2,6-bisphosphate stimulate lung phosphofructokinase. Half maximal stimulations occur in the range of 24.1-70.9 microM glucose 1,6-bisphosphate and 0.17-0.34 microM fructose 2,6-bisphosphate.