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     

Enzymatic preparation of high-specific-activity β-d-[6,6′-H]fructose-2,6-bisphosphate: Application to a sensitive assay for fructose-2,6-bisphosphatase

β-d-Fructose-2,6-bisphosphate (Fru-2,6-P₂) is an important regulator of eukaryotic glucose homeostasis, functioning as a potent activator of 6-phosphofructo-1-kinase and inhibitor of fructose-1,6-bisphosphatase. Pharmaceutical manipulation of intracellular Fru-2,6-P₂ levels, therefore, is of interest for the treatment of certain diseases, including diabetes and cancer. [2-³²P]Fru-2,6-P₂ has been the reagent of choice for studying the metabolism of this effector molecule; however, its short half-life necessitates frequent preparation. Here we describe a convenient, economical, one-pot enzymatic preparation of high-specific-activity tritium-labeled Fru-2,6-P₂. The preparation involves conversion of readily available, carrier-free d-[6,6′-³H]glucose to [6,6′-³H]Fru-2,6-P₂ using hexokinase, glucose-6-phosphate isomerase, and 6-phosphofructo-2-kinase. The key reagent in this preparation, bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from human liver, was produced recombinantly in Escherichia coli and purified in a single step using an appendant C-terminal hexa-His affinity tag. Following purification by anion exchange chromatography using triethylammonium bicarbonate as eluant, radiochemically pure [6,6′-³H]Fru-2,6-P₂ having a specific activity of 50 Ci/mmol was obtained in yields averaging 35%. [6,6′-³H]Fru-2,6-P₂ serves as a stable, high-specific-activity substrate in a facile assay capable of detecting fructose-2,6-bisphosphatase in the range of 10⁻¹⁴ to 10⁻¹⁵ mol, and it should prove to be useful in many studies of the metabolism of this important biofactor.     

6-Phosphofructo-2-kinase and D-fructose-2,6-bisphosphatase activities in mung bean seedlings

6-Phosphofructo-2-kinase (ATP: D-fructose-6-phosphate-2-phosphotransferase) and D-fructose-2,6-bisphosphatase activities have been found in extracts prepared from etiolated mung bean seedlings. The activity of 6-phosphofructo-2-kinase exhibits a sigmoidal shape in response to changes in concentrations of both substrates, D-fructose 6-phosphate and ATP (S_0.5 values of 1.8 and 1.2 mM, respectively). Inorganic orthophosphate (P_i) has a strong stimulating effect on the 2-kinase activity (A_0.5 at about 2 mM), moderately increasing the V_max and modifying the response into hyperbolic curves with K_m values of 0.4 and 0.2 mM for fructose 6-phosphate and ATP, respectively. 3-Phosphoglycerate (I_0.5 about 0.15 mM) partially inhibited the kinase activity by counteracting the P_i activation. In contrast, the activity of D-fructose-2,6-bisphosphatase (K_m 0.38 mM) is strongly inhibited by P_i (I_0.5 0.8 mM) lowering its affinity to fructose-2,6-P₂ (K_m 1.4 mM). 3-Phosphoglycerate activites the enzyme (A_0.5 at about 0.3 mM) without causing a significant change in its K_m for fructose-2,6-P₂. The activities of both of these enzymes in relationship to the metabolic role of D-fructose 2,6-bisphosphate in the germinating seed is discussed.     

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P₂) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P₂ were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P₂ had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-¹³C]glucose, in the lines possessing the highest amounts of Fru-2,6-P₂, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of ¹⁴C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P₂ can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates. Received: 29 March 2000 / Accepted: 13 June 2000     

Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by phenylglyoxal. Evidence for essential arginine residues.

Treatment of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with the arginine-specific reagent, phenylglyoxal, irreversibly inactivated both 6-phosphofructo-2-kinase and fructose-6-bisphosphatase in a time-dependent and dose-dependent manner. Fructose 6-phosphate protected against 2,6-phosphofructo-2-kinase inactivation, whereas MgGTP protected against fructose-2,6-bisphosphatase inactivation. Semi-logarithmic plots of the time course of inactivation by different phenylglyoxal concentrations were non-linear, suggesting that more than one arginine residue was modified. The stoichiometry of phenylglyoxal incorporation indicated that at least 2 mol/mol enzyme subunit were incorporated. Enzyme which had been phosphorylated by cyclic-AMP-dependent protein kinase was inactivated to a lesser degree by phenylglyoxal, suggesting that the serine residue (Ser32) phosphorylated by cyclic-AMP-dependent protein kinase interacts with a modified arginine residue. Chymotryptic cleavage of the modified protein and microsequencing showed that Arg225, in the 6-phosphofructo-2-kinase domain, was one of the residues modified by phenylglyoxal. The protection by fructose 6-phosphate against the labelling of chymotryptic fragments containing Arg225, suggests that this residue is involved in fructose 6-phosphate binding in the 6-phosphofructo-2-kinase domain of the bifunctional enzyme.     

Rat hepatoma (HTC) cell 6-phosphofructo-2-kinase differs from that in liver and can be separated from fructose-2,6-bisphosphatase

6-Phosphofructo-2-kinase was purified from rat liver and hepatoma (HTC) cells. The HTC cell enzyme had kinetic properties different from those of the liver enzyme (more sensitive to inhibition by citrate and not inhibited by sn-glycerol 3-phosphate) and was not a substrate of the cyclic-AMP-dependent protein kinase. Unlike the liver enzyme, which is bifunctional and phosphorylated by fructose 2,6-[2-32P]bisphosphate, the HTC cell enzyme contained no detectable fructose-2,6-bisphosphatase activity and phosphorylation by fructose 2,6-[2-32P]-bisphosphate could not be detected. HTC cell fructose-2,6-bisphosphatase could be separated from 6-phosphofructo-2-kinase activity by purification. Antibodies raised against liver 6-phosphofructo-2-kinase did not precipitate HTC cell fructose-2,6-bisphosphatase whose kinetic properties were completely different from those of the liver enzyme.     

Hormonal control of fructose 2,6-bisphosphate concentration in isolated rat hepatocytes.

The ability of glucagon and of adrenaline to affect the concentration of fructose 2,6-bisphosphate in isolated hepatocytes was re-investigated because of important discrepancies existing in the literature. We were unable to detect a significant difference in the sensitivity of the hepatocytes with regard to the effect of glucagon to initiate the interconversion of phosphorylase, pyruvate kinase, 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase, and also to cause the disappearance of fructose 2,6-bisphosphate. In contrast, we have observed differences in the time-course of these various changes, since the interconversions of phosphorylase and of pyruvate kinase were at least twice as fast as those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. When measured in a cell-free system in the presence of MgATP, the cyclic AMP-dependent interconversion of pyruvate kinase was 5-10-fold more rapid than those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. These data indicate that 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase are relatively poor substrates for cyclic AMP-dependent protein kinase; they also support the hypothesis that the two catalytic activities belong to a single protein. Adrenaline had only a slight effect on the several parameters under investigation, except for the activation of phosphorylase. In the absence of Ca2+ ions from the incubation medium, however, adrenaline had an effect similar to that of glucagon.     

Lysine 356 is a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Lysine 356 has been implicated by protein modification studies as a fructose-2,6-bisphosphate binding site residue in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Kitajima, S., Thomas, H., and Uyeda, K. (1985) J. Biol. Chem. 260, 13995-14002). However, Lys-356 is found in the fructose-2,6-bisphosphatase domain (Bazan, F., Fletterick, R., and Pilkis, S. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). In order to ascertain whether Lys-356 is involved in fructose-2,6-bisphosphatase catalysis and/or domain/domain interactions of the bifunctional enzyme, Lys-356 was mutated to Ala, expressed in Escherichia coli, and then purified to homogeneity. Circular dichroism experiments indicated that the secondary structure of the Lys-356-Ala mutant was not significantly different from that of the wild-type enzyme. The Km for fructose 2,6-bisphosphate and the Ki for the noncompetitive inhibitor, fructose 6-phosphate, for the fructose-2,6-bisphosphatase of the Lys-356-Ala mutant were 2700- and 2200-fold higher, respectively, than those of the wild-type enzyme. However, the maximal velocity and the Ki for the competitive product inhibitor, inorganic phosphate, were unchanged compared to the corresponding values of the wild-type enzyme. Furthermore, in contrast to the wild-type enzyme, which exhibits substrate inhibition, there was no inhibition by substrate of the Lys-356-Ala mutant. In the presence of saturating substrate, inorganic phosphate, which acts by relieving fructose-6-phosphate and substrate inhibition, is an activator of the bisphosphatase. The Ka for inorganic phosphate of the Lys-356-Ala mutant was 1300-fold higher than that of the wild-type enzyme. The kinetic properties of the 6-phosphofructo-2-kinase of the Lys-356-Ala mutant were essentially identical with that of the wild-type enzyme. The results demonstrate that: 1) Lys-356 is a critical residue in fructose-2,6-bisphosphatase for binding the 6-phospho group of fructose 6-phosphate/fructose 2,6-bisphosphate; 2) the fructose 6-phosphate binding site is responsible for substrate inhibition; 3) Inorganic phosphate activates fructose-2,6-bisphosphatase by competing with fructose 6-phosphate for the same site; and 4) Lys-356 is not involved in 6-phosphofructo-2-kinase substrate/product binding or catalysis.     

Contribution of different protein phosphatases to the dephosphorylation of 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat liver.

The nature of rat liver protein phosphatases involved in the dephosphorylation of the glycolytic key enzyme 6-phosphofructo-1-kinase and the regulatory enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was investigated. In terms of the classification system proposed by Ingebritsen & Cohen [(1983) Eur. J. Biochem. 132, 255-261], only the type-2 protein phosphatases 2A (which can be separated into 2A1 and 2A2) and 2C act on these substrates. Fractionation of rat liver extracts by anion-exchange chromatography and gel filtration revealed that protein phosphatase 2A is responsible for most of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase phosphatase activity (activity ratio 2A/2C = 4:1). On the other hand, 6-phosphofructo-1-kinase phosphatase activity is equally distributed between protein phosphatases 2A (2A1 plus 2A2) and 2C. In addition, the possible role of low-Mr compounds for the control of purified protein phosphatase 2C was examined. At near-physiological concentrations, none of the metabolites studied significantly affected the rate of dephosphorylation of 6-phosphofructo-1-kinase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, pyruvate kinase or fructose-1,6-bisphosphatase.     

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.     

Insulin-mediated regulation of heart atrial and ventricular 6-phosphofructo-1-kinase

Atrial 6-phosphofructo-1-kinase activity from the hearts of diabetic rats was decreased by 50%, but ventricular 6-phosphofructo-1-kinase activity was found not to be insulin-sensitive. This decrease in atrial 6-phosphofructo-1-kinase activity during diabetes was characterized by diminished levels of all three types of 6-phosphofructo-1-kinase subunits. As shown by immunological titration and column chromatography, the population of native 6-phosphofructo-1-kinase isozymes in the ventricles was not measurably affected during insulin deprivation. However, the atrial isozyme population in diabetic rat heart appeared to contain, on a relative basis, higher levels of the isozymic forms containing the L-type subunit. Measurement of the levels of this subunit indicated that in diabetic atria it was less affected than the other subunits. In the ventricles, insulin deficiency did not promote significant losses of fructose-2,6-P2; but, in diabetic rats, the atrial levels of this activator were decreased by 80% and subsequently restored by insulin treatment. These data suggest that any insulin-mediated effects on ventricular 6-phosphofructo-1-kinase activity and resultant effects on ventricular glycolysis do not appear to be exerted through changes in enzyme concentration, but probably through changes in modulators other than fructose-2,6-P2. In contrast to the ventricles, it appears that insulin exerts its effects on atrial 6-phosphofructo-1-kinase activity and, in part, influences atrial glycolysis through alteration of fructose-2,6-P2 levels, enzyme concentration, and isozymic content.     

The Effect of Elevated Concentrations of Fructose 2,6-Bisphosphate on Carbon Metabolism during Deacidification in the Crassulacean Acid Metabolism Plant Kalanchöe daigremontiana

In C₃ plants, the metabolite fructose 2,6-bisphosphate (Fru 2,6-P₂) has an important role in the regulation of carbon partitioning during photosynthesis. To investigate the impact of Fru 2,6-P₂ on carbon metabolism during Crassulacean acid metabolism (CAM), we have developed an Agrobacterium tumefaciens-mediated transformation system in order to alter genetically the obligate CAM plant Kalanchöe daigremontiana. To our knowledge, this is the first report to use genetic manipulation of a CAM species to increase our understanding of this important form of plant metabolism. Transgenic plants were generated containing a modified rat liver 6-phosphofructo-2-kinase gene. In the plants analyzed the activity of 6-phosphofructo-2-kinase ranged from 175% to 198% of that observed in wild-type plants, resulting in Fru 2,6-P₂ concentrations that were 228% to 350% of wild-type plants after 2 h of illumination. A range of metabolic measurements were made on these transgenic plants to investigate the possible roles of Fru 2,6-P₂ during Suc, starch, and malic acid metabolism across the deacidification period of CAM. The results suggest that Fru 2,6-P₂ plays a major role in regulating partitioning between Suc and starch synthesis during photosynthesis. However, alterations in Fru 2,6-P₂ levels had little effect on malate mobilization during CAM fluxes.     

Kinetic properties of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves.

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa. The purified protein had activity of both 6-phosphofructo-2-kinase specific activity 10.4-15.9 nmol min(-1) x mg protein (-1) and fructose-2,6-bisphosphatase (specific activity 1.65-1.75 nmol x mol(-1) mg protein(-1). The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate, and inhibited by 3-carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate, 3-phosphoglycerate was a mixed inhibitor with respect to both fructose 6-phosphate and ATP. Fructose-2,6-bisphosphatase activity was sensitive to product inhibition; inhibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6-phosphate was mixed. These kinetic properties support the view that the level of fructose 2,6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities of the bifunctional enzyme.     

Fructose 2,6-bisphosphate and 6-phosphofructo-2-kinase during liver regeneration.

Glycogen and fructose 2,6-bisphosphate levels in rat liver decreased quickly after partial hepatectomy. After 7 days the glycogen level was normalized and fructose 2,6-bisphosphate concentration still remained low. The 'active' (non-phosphorylated) form of 6-phosphofructo-2-kinase varied in parallel with fructose 2,6-bisphosphate levels, whereas the 'total' activity of the enzyme decreased only after 24 h, similarly to glucokinase. The response of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from hepatectomized rats (96 h) to sn-glycerol 3-phosphate and to cyclic AMP-dependent protein kinase was different from that of the enzyme from control animals and similar to that of the foetal isoenzyme.     

Induction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA by refeeding and insulin

The effects of fasting/refeeding and untreated or insulin-treated diabetes on the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its mRNA in rat liver were determined. Both enzymatic activities fell to 20% of control values with fasting or streptozotocin-induced diabetes and were coordinately restored to normal within 48 h of refeeding or 24 h of insulin administration. These alterations in enzymatic activities were always mirrored by corresponding changes in amount of enzyme as determined by phosphoenzyme formation and immunoblotting. In contrast, mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase did not decrease during starvation or in diabetes, but there was a 3-6-fold increase upon refeeding a high carbohydrate diet to starved rats or insulin treatment of diabetic rats. The decrease of the enzyme in starved or diabetic rats without associated changes in mRNA levels suggests a decrease in the rate of mRNA translation, an increase in enzyme degradation, or both. The rise in enzyme amount and mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with refeeding and insulin treatment suggests an insulin-dependent stimulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression. Northern blots of RNA from heart, brain, kidney, and skeletal muscle probed with restriction fragments of a full-length cDNA from liver showed that only skeletal muscle contained an RNA species that hybridized to any of the probes. Skeletal muscle mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was 2.0 kilobase pairs but in contrast to the liver message (2.2 kilobase pairs) was not regulated by refeeding.     

Synthesis and degradation of fructose 2,6-bisphosphate in endosperm of castor bean seedlings

The aim of this work was to examine the possibility that fructose 2,6-bisphosphate (Fru-2,6-P₂) plays a role in the regulation of gluconeogenesis from fat. Fru-2,6-P₂ is known to inhibit cytoplasmic fructose 1,6-bisphosphatase and stimulate pyrophosphate:fructose 6-phosphate phosphotransferase from the endosperm of seedlings of castor bean (Ricinus communis). Fru-2,6-P₂ was present throughout the seven-day period in amounts from 30 to 200 picomoles per endosperm. Inhibition of gluconeogenesis by anoxia or treatment with 3-mercaptopicolinic acid doubled the amount of Fru-2,6-P₂ in detached endosperm. The maximum activities of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase (enzymes that synthesize and degrade Fru-2,6-P₂, respectively) were sufficient to account for the highest observed rates of Fru-2,6-P₂ metabolism. Fructose 6-phosphate,2-kinase exhibited sigmoid kinetics with respect to fructose 6-phosphate. These kinetics became hyperbolic in the presence of inorganic phosphate, which also relieved a strong inhibition of the enzyme by 3-phosphoglycerate. Fructose 2,6-bisphosphatase was inhibited by both phosphate and fructose 6-phosphate, the products of the reaction. The properties of the two enzymes suggest that in vivo the amounts of fructose-6-phosphate, 3-phosphoglycerate, and phosphate could each contribute to the control of Fru-2,6-P₂ level. Variation in the level of Fru-2,6-P₂ in response to changes in the levels of these metabolites is considered to be important in regulating flux between fructose 1,6-bisphosphate and fructose 6-phosphate during germination.     

The yeast FBP26 gene codes for a fructose-2,6-bisphosphatase.

Sequencing of an open reading frame 450 bp downstream from the yeast VPS35 gene revealed a putative peptide of 452 amino acids and 52.7 kDa. The predicted amino acid sequence has 45% identity with the 55-kDa subunit of the 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase (EC 2.7.1.105/EC 3.1.3.46) from rat liver and 42% identity with 480 amino acids in the center of the recently reported 93.5-kDa subunit of yeast 6-phosphofructo-2-kinase (EC 2.7.1.105). The product of the new yeast gene is similar to the entire sequence of the bifunctional rat liver enzyme and, unlike yeast 6-phosphofructo-2-kinase, has the histidine residue essential for fructose-2,6-bisphosphatase activity. Extracts from a chromosomal null mutant strain, fbp26::HIS3, incubated in the presence of [2-32P]fructose 2,6-P2, lacked in autoradiograms the characteristic 56-kDa labeled band observed in wild-type. The same band was intensified 3-fold over wild-type level with the FBP26 gene introduced on multicopy in the fbp26::HIS3 background. A similar increase was found for fructose-2,6-bisphosphatase activity in the same extracts. The FBP26 gene did not cause detectable increase in 6-phosphofructo-2-kinase activity when introduced on multicopy in a pfk26::LEU2 mutant, indicating that its gene product is predominantly a fructose-2,6-bisphosphatase. Growth on glucose, fructose, galactose, pyruvate, and glycerol/lactate was not impaired in strains carrying the fbp26::HIS3 allele.