Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [gamma-32P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-Km (0.1 microM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 microM) and stimulated 2-3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.     

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.     

Evidence for the short-term regulation of glycolytic flux in the isolated perfused ventricle of the land snail Helix lucorum (L.) after treatment with serotonin (5-hydroxytryptamine)

The glycolytic flux and the regulation of phosphofructokinase (PFK) activity by fructose 2,6-bisphosphate and covalent modification was investigated in isolated ventricles of land snail Helix lucorum perfused with or without serotonin. Serotonin evoked a significant increase in the level of glycolytic intermediates and a threefold increase of glycolytic flux. Studies of saturation curves of PFK for the substrate fructose 6-phosphate at pH similar to intracellular pH of heart muscle showed that serotonin increases enzyme sensitivity to activation by fructose 6-phosphate. Moreover, PFK preparations from ventricles perfused with serotonin exhibited lower K _a values for the activators AMP and fructose 2,6-bisphosphate, compared with the enzyme preparations from serotonin-untreated ventricles. The results suggest that PFK was converted to a more active form when exposed to serotonin. In vitro experiments of PFK phosphorylation showed that the conversion of the enzyme to a more active form was possibly due to its phosphorylation by an endogenous cyclic-AMP-dependent protein kinase. The concentration of fructose 2,6-bisphosphate increased in serotonin-treated ventricles and it exerted a synergistic effect with AMP on the activation of PFK. The bound fraction of glycolytic enzymes increased in the serotonin-treated ventricles only after the 4th min of perfusion. The results suggest that the stimulation of glycolytic flux in the ventricles of H. lucorum in the first minutes of perfusion with serotonin was partly due to the activation of PFK via enzyme molecule covalent modification and to increase of fructose 2,6-bisphosphate. Accepted: 8 April 1997     

Occurrence and characterization of fructose 6-phosphate, 2-kinase and fructose 2,6-bisphosphatase in Euglena gracilis

1. Fructose 6-phosphate, 2-kinase and fructose 2,6-bisphosphatase occurred in Euglena gracilis SM-ZK, and is located in cytosol. 2. Fructose 6-phosphate, 2-kinase and fructose 2,6-bisphosphatase were partially purified, and both enzyme activities were not separated during the partial purification. 3. The pH optimum for fructose 6-phosphate, 2-kinase activity was 7.0. The saturation curve of the enzyme activity for ATP concentration was hyperbolic, and the Km value for the substrate was 0.88 mM. On the other hand, the saturation curve of the enzyme activity for fructose 6-phosphate concentration was sigmoidal, and the K0.5 value for the substrate was 70 microM. 4. The pH optimum for fructose 2,6-bisphosphatase activity was 6.5. The saturation curve for fructose 2,6-bisphosphate concentration was sigmoidal, and the K0.5 value for the substrate was 1.29 microM. Fructose 2,6-bisphosphate showed a substrate inhibition at high concentration over 5 microM, and the enzyme activity was completely inhibited by 20 microM of fructose 2,6-bisphosphate.     

The purification, characterization and regulatory properties of liver phosphofructokinase in the Arabian one-humped camel (Camelus dromedarius)

1. Phosphofructokinase from camel liver was purified to homogeneity more than 3600-fold, and the yield of the preparation was 46%. 2.The sodium dodecyl sulphate-treated purified enzyme migrated as a single band in 10% polyacrylamide gel. 3. The enzyme is a tetramer, with a monomer Mr 90,000. 4. The regulatory properties of the purified enzyme from camel liver were studied at pH 7.0. 5. The enzyme displayed cooperativity with respect to fructose 6-phosphate and was inhibited by high concentrations of ATP. 6. The enzyme was also inhibited by citrate, phosphocreatine and 2,3-bisphosphoglycerate. 7. On the other hand, ADP, AMP, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate were all found to be strong activators for camel liver phosphofructokinase.     

Effect of experimental hypothyroidism on the control of 6-phosphofructo-1-kinase activity in rat jejunal mucosa.

Changes in the activity of 6-phosphofructo-1-kinase (PFK, EC 2.7.1.11) from the epithelial cells of rat small intestine during experimental hypothyroidism were studied. Hypothyroidism resulted in significant decreases in the plasma concentrations of total tri-iodothyronine, free tri-iodothyronine, total thyroxine, free thyroxine and insulin. These changes were associated with a significant increase in the plasma concentration of thyrotropin. The total activity and activity ratios (activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (v0.5/V)) of jejunal PFK of hypothyroid rats were significantly diminished as compared to control rats. PFK of hypothyroid rats was more sensitive to inhibition by ATP. The mucosal enzyme of both control and hypothyroid state was sensitive to stimulation by AMP and fructose 2,6-bisphosphate. It is concluded that during hypothyroidism the rate of glycolytic pathway in the small intestine is reduced as a result of a fall in glucose uptake, and the subsequent kinetic changes of PFK are primarily to maintain the concentrations of fructose 6-phosphate (and glucose 6-phosphate) during the reduced glycolytic flux. These changes in PFK activity may be caused by changes in plasma insulin concentrations, glucose utilization and fructose 2,6-bisphosphate concentrations.     

Fructose-2,6-bisphosphatase and 6-phosphofructo-2-kinase are separable in yeast.

Fructose-2,6-bisphosphatase was purified from yeast and separated from 6-phosphofructo-2-kinase and alkaline phosphatase. The enzyme released Pi from the 2-position of fructose 2,6-bisphosphate and formed fructose 6-phosphate in stoichiometric amounts. The enzyme displays hyperbolic kinetics towards fructose 2,6-bisphosphate, with a Km value of 0.3 microM. It is strongly inhibited by fructose 6-phosphate. The inhibition is counteracted by L-glycerol 3-phosphate. Phosphorylation of the enzyme by cyclic-AMP-dependent protein kinase causes inactivation, which is reversible by the action of protein phosphatase 2A.     

Regulation of 6-phosphofructo-1-kinase from the epithelial cells of rat small intestine during pregnancy and lactation

The regulation of 6-phosphofructo-1-kinase (PFK) in the epithelial cells of rat small intestine was studied during pregnancy and lactation. The total activities and activity ratios (activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (nu 0.5/V] of the partially purified mucosal PFK were found to increase initially in early pregnant rats (11-12 days of gestation) and to fall back to normal in late pregnant rats (19-20 days of gestation). These changes in enzyme activity during pregnancy were associated with similar changes in the circulating levels of progesterone. The maximal activity and activity ratio (nu 0.5/V) were increased in male and female rats injected with progesterone. An increase in the total activity and activity ratio of mucosal PFK was also obtained in lactating rats. However, the enzyme was not strongly activated by inorganic phosphate, fructose 2,6-bisphosphate or glucose 1,6-bisphosphate either in early pregnant or lactating rats. These results indicate that mucosal PFK is already present as an active form during early pregnancy and lactation. Therefore, it is suggested that female sex hormones increase the circulating levels of insulin during early pregnancy which, in turn, positively affect the activity of mucosal PFK which could be also stimulated by the increased levels of fructose 2,6-bisphosphate. The increased activity of PFK in the peak lactating rats could be possible because of an increased demand for lactate production from glucose together with the stimulation of PFK by the increased concentrations of fructose 2,6-bisphosphate which therefore increases the rate of glycolysis.     

Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana ( Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (alpha) and 60-kDa (beta) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi ( K(m) values = 32, 9.7, 25, and 410 microM, respectively) in the presence of saturating (5 microM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity ( K(a)=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5-6.0 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP ( K(m)=34 and 21 microM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P ( S0.5=540 and 90 microM, respectively). Allosteric effects of phospho enolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II ( I50=2.3 and 900 microM, respectively), while Pi activated PFK I by reducing its sensitivity to PEP inhibition. Our results are consistent with: (i) the respiratory climacteric being regulated by fine (allosteric) control of pre-existing enzymes; and (ii) primary and secondary glycolytic flux control being exerted at the levels of PEP and Fru 6-P metabolism, respectively.     

Purification and characterization of fructose-2,6-bisphosphatase, a substrate-specific cytosolic enzyme from leaves

Fructose-2,6-bisphosphatase (EC 3.1.3.46), which hydrolyzes fructose 2,6-bisphosphate to fructose 6-phosphate and Pi, has been purified to apparent homogeneity from spinach leaves and found to be devoid of fructose-6-phosphate,2-kinase activity. The isolated enzyme is a dimer (76 kDa determined by gel filtration) composed of two 33-kDa subunits. The enzyme is highly specific and displays hyperbolic kinetics with its fructose 2,6-bisphosphate substrate (Km = 32 microM). The products of the reaction, fructose 6-phosphate and Pi, along with AMP and Mg2+ are inhibitors of the enzyme. Nonaqueous cell fractionation revealed that, like the fructose 2,6-bisphosphate substrate, fructose-2,6-bisphosphatase as well as fructose-6-phosphate,2-kinase occur in the cytosol of spinach leaves.     

Regulation of rat liver fructose 2,6-bisphosphatase

An enzyme activity that catalyzes the hydrolysis of phosphate from the C-2 position of fructose 2,6-bisphosphate has been detected in rat liver cytoplasm. The S0.5 for fructose 2,6-bisphosphate was about 15 microM and the enzyme was inhibited by fructose 6-phosphate (Ki 40 microM) and activated by Pi (KA 1 mM). Fructose 2,6-bisphosphatase activity was purified to homogeneity by specific elution from phosphocellulose with fructose by specific elution from phosphocellulose with fructose 6-phosphate and had an apparent molecular weight of about 100,000, 6-phosphofructo 2-kinase activity copurified with fructose 2,6-bisphosphatase activity at each step of the purification scheme. Incubation of the purified protein with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in the incorporation of 1 mol of 32P/mol of enzyme subunit (Mr = 50,000). Concomitant with this phosphorylation was an activation of the fructose 2,6-bisphosphatase and an inhibition of the 6-phosphofructo 2-kinase activity. Glucagon addition to isolated hepatocytes also resulted in an inhibition of 6-phosphofructo 2-kinase and activation of fructose 2,6-bisphosphatase measured in cell extracts, suggesting that the hormone regulates the level of fructose 2,6-bisphosphate by affecting both synthesis and degradation of the compound. These findings suggest that this enzyme has both phosphohydrolase and phosphotransferase activities i.e. that it is bifunctional, and that both activities can be regulated by cAMP-dependent phosphorylation.     

Fructose-2,6-bisphosphatase from rat liver

An enzyme that catalyzes the stoichiometric conversion of fructose 2,6-bisphosphate into fructose 6-phosphate and inorganic phosphate has been purified from rat liver. This fructose 2,6-bisphosphatase copurified with phosphofructokinase 2 (ATP: D-fructose 6-phosphate 2-phosphotransferase) in the several separation procedures used. The enzyme was active in the absence of Mg2+ and was stimulated by triphosphonucleotides in the presence of Mg2+ and also by glycerol 3-phosphate, glycerol 2-phosphate and dihydroxyacetone phosphate. It was strongly inhibited by fructose 6-phosphate at physiological concentrations and this inhibition was partially relieved by glycerol phosphate and dihydroxyacetone phosphate. The activity of fructose 2,6-bisphosphatase was increased severalfold upon incubation in the presence of cyclic-AMP-dependent protein kinase and cyclic AMP. The activation resulted from an increase in V (rate at infinite concentration of substrate) and from a greater sensitivity to the stimulatory action of ATP and of glycerol phosphate at neutral pH. The activity of fructose 2,6-bisphosphatase could also be measured in crude liver preparations and in extracts of hepatocytes. It was then increased severalfold by treatment of the cells with glucagon, when measured in the presence of triphosphonucleotides.     

Regulation of glycolysis during acclimation of scallops (Patinopecten yessoensis Jay) to anaerobiosis

Some glycolytic metabolites in the adductor muscle were measured after transfer of scallops from aerobic to anaerobic saltwater for 12 h. The level of octopine increased gradually during the initial 3 h incubation, and thereafter the level increased rapidly up to 12 h. The ATP level also did not show any significant change for the initial 3 h, and then decreased rapidly. The fructose 2,6-biphosphate (Fru 2,6-BP) level increased drastically during the initial 3 h incubation, but thereafter the level did not show any significant change up to 12 h. In the short-term effects of anaerobiosis for 90 min, the level of fructose 6-phosphate (Fru 6-P) increased just after transfer to anaerobiosis, and then its level decreased. In contrast, the fructose 1,6-biphosphate (Fru 1,6-BP) level increased greatly, at the time when both glucose 6-phosphate (Glc 6-P) and Fru 6-P decreased. The Fru 2,6-BP level did not any significant change during the initial 15 min incubation, but thereafter the level increased gradually up to 90 min. Scallop 6-phosphofructo 1-kinase (EC 2.7.1.11) (PFK1) was strongly activated by 1 microM Fru 2,6-BP when 0.2 mM Fru 6-P was used as a substrate, but the activity was not affected at 5 mM Fru 6-P. In view of these results, the regulation mechanism of glycolysis is discussed.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification,kinetics and immunological properties

Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mol · l^-1). The inhibition was counteracted by inorganic phosphate and, particularly, by glycerol 3-phosphate. In the presence of inorganic phosphate and glycerol 3-phosphate the frog muscle fructose-2,6-bisphosphatase was much more sensitive to fructose 6-phosphate inhibition than was the rat M-type fructose-2,6-bisphosphatase. No change in kinetics and no phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was observed after incubation with protein kinase C and a Ca²⁺/calmodulin-dependent protein kinase. The kinetics of frog muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, although they would favour an initial increase in fructose 2,6-bisphosphate in exercising frog muscle, cannot fully account for the changes in fructose 2,6-bisphosphate observed in muscle of exercising frog. Regulatory mechanisms not yet studied must be involved in working frog muscle in vivo.Abbreviations BSA bovine serum albumin - Ca/CAMK Ca²⁺/calmodulin-dependent protein kinase (EC 2.7.1.37) - CL anti-l-type PFK-21 FBPase-2 antiserum - DTT dithiothreitol - EP phosphorylated enzyme intermediate - FBPase-2 fructose-2,6-bisphosphatase (EC 3.1.3.46) - F2,6P₂ fructose 2,6-bisphosphate - I_0,5 inhibitor concentration required to decrease enzyme activity by 50% - MCL-2 anti-PFK-2/FBPase-2 antiserum - M_r relative molecular mass - PEG polyethylene glycol - PFK-1 6-phosphofructo-1-kinase (EC 2.7.1.11) - PKF-2 6-phosphofructo-2-kinase (EC 2.7.1.105) - PKA protein kinase A = cyclic AMP-dependent protein kinase (EC 2.7.1.37) - PKC protein kinase C (EC 2.7.1.37) - SDS sodium dodecylsulphate - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - U unit of enzyme activity     

Allosteric properties of phosphofructokinase from the epithelial cells of thermally injured rat small intestine

1. The allosteric properties of phosphofructokinase from the epithelial cells of thermally injured rat small intestine were studied and compared with those properties of the normal rats. 2. The fructose 6-phosphate saturation curve of mucosal phosphofructokinase from thermally injured rats (3 days post injury, 33% of body surface area) displayed cooperatively; the ratio of the activity observed at pH 7.0 in the presence of 0.5 mM fructose 6-phosphate and 2.5 mM-ATP to the optimal activity at pH 8.0, v 0.5/V, was 0.42 +/- 0.02 in the normal rats and 0.22 +/- 0.03 in the injured rats. 3. The enzyme from thermally injured rats was very sensitive to inhibition by ATP as compared to that from normal rats. 4. The enzyme from thermally injured rats was inhibited by citrate and phosphocreatine in a synergistic manner with ATP. 5. Activation under nearly cellular conditions was produced by ADP, AMP and glucose-1,6-biphosphate. 6. In general, the mucosal enzyme of thermally injured rats was more susceptible to inhibition or activation by various metabolites than the enzyme of the normal rats. 7. These results may suggest that mucosal phosphofructokinase of thermally injured rats may not be subject to the same control mechanism as the normal rats in vivo due to changes in the concentrations of fructose-2,6-biphosphate.     

On the mechanism by which noradrenaline increases the activity of phosphofructokinase in isolated rat adipocytes.

We confirmed that, as reported by Sooranna & Saggerson [(1982) Biochem. J. 202, 753-758], the affinity of 6-phosphofructo-1-kinase (PFK) for fructose 6-phosphate in an adipocyte extract was increased after incubation of the cells in the presence of noradrenaline. The participation of fructose 2,6-bisphosphate in this kinetic modification could be excluded, because the noradrenaline effect persisted after extensive gel filtration of the extracts and also because the treatment did not cause any change in the concentration of fructose 2,6-bisphosphate in the adipocytes. Oleic acid was found to be another potent positive effector of PFK in an adipocyte extract, with a Ka of 10 microM. Its effect was synergistic with that of fructose 2,6-bisphosphate and AMP, and was counteracted by serum albumin. Palmitic acid had a similar effect. We conclude that the large increase in fatty acid concentration caused by noradrenaline treatment is an explanation for the activation of phosphofructokinase at low fructose 6-phosphate concentrations in an adipocyte extract.     

ATP-dependent activation of a new form of spinach leaf 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase

A novel form of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase that possesses little 2-kinase or bisphosphatase activity as isolated has been partially purified from spinach (Spinacia oleracea L.) leaves. However, the new form can be activated by pretreatment with Mg X ATP at room temperature. After ATP activation, the fructose 2,6-bisphosphatase activity has a Michaelis constant for fructose 2,6-bisphosphate of about 1 mM, and is inhibited by high substrate concentrations (greater than 2 mM) and both end products. The kinase/phosphatase activity ratio of the new form was dependent on pH and varied from 0.3 at pH 7.0 to 5.0 at pH 8.2. In contrast, the previously characterized form of the enzyme (which is isolated in an active form and is unaffected by preincubation with Mg X ATP) had an activity ratio of about 2 that was insensitive to pH over the range tested. The ATP-dependent activation of the new enzyme form was stimulated by fructose 6-phosphate and inhibited by glucose 6-phosphate. These results explain why activation is not observed during assay of this enzyme, and indicate that the activation process may be regulated by metabolites. Collectively, these data provide further evidence for the existence, in spinach leaves, of two molecular forms of the enzyme which exhibit different kinetic properties.     

The subcellular location and characteristics of pyrophosphate-fructose-6-phosphate 1-phosphotransferase from suspension-cultured cells of soybean

The cytoplasm was identified as the probable location of pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) in suspension-cultured cells of soybean (Glycine max L.). The characteristics of the partially purified enzyme were investigated. The activity was strongly dependent on the presence of fructose 2,6-bisphosphate and this activator exerted its effects through a dramatic increase in the affinity of the enzyme for its substrates, fructose 6-phosphate and inorganic pyrophosphate. Saturation curves for all substrates were hyperbolic. The apparent molecular weight of the partially purified enzyme was 183000 by gel filtration chromatography and 128000 by sucrose-density-gradient centrifugation. The activation by fructose 2,6-bisphosphate was not accompanied by any measurable change in molecular weight. The possible role of this enzyme in the metabolism of non-photosynthetic sink tissues is discussed.Abbreviations PFP pyrophosphate-fructose-6-phosphate 1-phosphotransferase - P_i phosphate - PP_i pyrophosphate     

Posttranslational Modification of 6-Phosphofructo-1-Kinase in Aspergillus niger

Two different enzymes exhibiting 6-phosphofructo-1-kinase (PFK1) activity were isolated from the mycelium of Aspergillus niger: the native enzyme with a molecular mass of 85 kDa, which corresponded to the calculated molecular mass of the deduced amino acid sequence of the A. niger pfkA gene, and a shorter protein of approximately 49 kDa. A fragment of identical size also was obtained in vitro by the proteolytic digestion of the partially purified native PFK1 with proteinase K. When PFK1 activity was measured during the proteolytic degradation of the native protein, it was found to be lost after 1 h of incubation, but it was reestablished after induction of phosphorylation by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the system. By determining kinetic parameters, different ratios of activities measured at ATP concentrations of 0.1 and 1 mM were detected with fragmented PFK1, as with the native enzyme. Fructose-2,6-biphosphate significantly increased the V_max of the fragmented protein, while it had virtually no effect on the native protein. The native enzyme could be purified only from the early stages of growth on a minimal medium, while the 49-kDa fragment appeared later and was activated at the time of a sudden change in the growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sequential purifications of PFK1 enzymes by affinity chromatography during the early stages of the fungal development suggested spontaneous posttranslational modification of the native PFK1 in A. niger cells, while from the kinetic parameters determined for both isolated forms it could be concluded that the fragmented enzyme might be more efficient under physiological conditions.     

Comparison of phosphofructokinases in submandibular glands of immature and adult rats

1. Phosphofructokinases (PFKs) in immature and adult rat submandibular glands were purified to near homogeneity, and their properties were compared. 2. PFK in immature gland was less sensitive to inhibition by ATP than adult PFK. 3. Saturation curve for fructose 6-phosphate of PFK in immature gland was less sigmoidal than that of adult PFK indicating the lower cooperativity of subunits in immature PFK. 4. Fructose 2,6-bisphosphate relieved PFK from inhibition by ATP in adult gland, but a similar effect was not clearly observed in immature gland PFK. 5. Adult PFK was a heterotetramer consisting of C-, M-, L-subunits, but in immature PFK another type of subunit, which was slightly smaller than L-subunit, existed in addition to C-, M- and L-subunits.