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.     

Effect of benzoate on the metabolism of fructose 2,6-bisphosphate in yeast

When benzoate (2 mM, pH 3.5) was added together with glucose (0.1 M) to a suspension of Saccharomyces cerevisiae in the stationary phase, it caused a relative increase in the concentration of glucose 6-phosphate and fructose 6-phosphate and a decrease in the concentration of fructose 1,6-bisphosphate. These effects are in confirmation of similar observations made by Krebs et al. [Biochem. J. 214, 657-663 (1983)] and are indicative of an inhibition of 6-phosphofructo-1-kinase. Benzoate also caused an about fourfold relative decrease in the concentration of fructose 2,6-bisphosphate, an increase in that of cyclic AMP with no change in that of ATP. It also greatly decreased the activation of 6-phosphofructo-2-kinase, but not that of trehalase, both of which normally occur upon addition of glucose to a yeast suspension. When added 10 min after glucose, benzoate caused a rapid (within 2-3 min) decrease in fructose 2,6-bisphosphate concentration and in 6-phosphofructo-2-kinase activity. In the presence of benzoate, there was also a parallel decrease in the concentration of fructose 2,6-bisphosphate and in the rate of ethanol production when the external pH was dropped from 5.0 to 2.5, with minimal change in the concentration of ATP. Purified 6-phosphofructo-2-kinase was inhibited by benzoate and also by an acid pH. Experiments with cell-free extracts did not provide an explanation for the rapid disappearance of fructose-2,6-bisphosphate or the inactivation of 6-phosphofructo-2-kinase in yeast upon addition of benzoate.     

Role of fructose 2,6-bisphosphate in the regulation of glycolysis and gluconeogenesis in chicken liver

Glucagon and dibutyryl cyclic AMP inhibited glucose utilization and lowered fructose 2,6-bisphosphate levels of hepatocytes prepared from fed chickens. Partially purified preparations of chicken liver 6-phosphofructo-1-kinase and fructose 1,6-bisphosphatase were activated and inhibited by fructose 2,6-bisphosphate, respectively. The sensitivities of these enzymes and the changes observed in fructose 2,6-bisphosphate levels are consistent with an important role for this allosteric effector in hormonal regulation of carbohydrate metabolism in chicken liver. In contrast, oleate inhibition of glucose utilization by chicken hepatocytes occurred without change in fructose, 2,6-bisphosphate levels. Likewise, pyruvate inhibition of lactate gluconeogenesis in chicken hepatocytes cannot be explained by changes in fructose 2,6-bisphosphate levels. Exogenous glucose caused a marked increase in fructose 2,6-bisphosphate content of hepatocytes from fasted but not fed birds. Both glucagon and lactate prevented this glucose effect. Fasted chicken hepatocytes responded to lower glucose concentrations than fasted rat hepatocytes, perhaps reflecting the species difference in hexokinase isozymes.     

The permissive effects of glucocorticoid on hepatic gluconeogenesis. Glucagon stimulation of glucose-suppressed gluconeogenesis and inhibition of 6-phosphofructo-1-kinase in hepatocytes from fasted rats

Production of [14C]glucose from [14C]lactate in the perfused livers of 24-h fasted adrenalectomized rats was not stimulated by 1 nM glucagon but was significantly increased by 10 nM hormone. Crossover analysis of glycolytic intermediates in these livers revealed a significant reduction in glucagon action at site(s) between fructose 6-phosphate and fructose 1,6-bisphosphate as a result of adrenalectomy. Site(s) between pyruvate and P-enolpyruvate was not affected. In isolated hepatocytes, adrenalectomy reduced glucagon response in gluconeogenesis while not affecting glucagon inactivation of pyruvate kinase. A distinct lack of glucagon action on 6-phosphofructo-1-kinase activity was noted in these cells. When hepatocytes were incubated with 30 mM glucose, lactate gluconeogenesis was greatly stimulated by glucagon. A reduction in both sensitivity and responsiveness to the hormone in gluconeogenesis was seen in the adrenalectomized rat. These changes were well correlated with similar impairment in glucagon action on 6-phosphofructo-1-kinase activity and fructose 2,6-bisphosphate content in hepatocytes from adrenalectomized rats incubated with 30 mM glucose. These results suggest that adrenalectomy impaired the gluconeogenic action of glucagon in livers of fasted rats at the level of regulation of 6-phosphofructo-1-kinase and/or fructose 2,6-bisphosphate content.     

Fructose 2,6-bisphosphate and its phosphorothioate analogue. Comparison of their hydrolysis and action on glycolytic and gluconeogenic enzymes.

Purified chicken liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was phosphorylated either from fructose 2,6-bis[2-32P]phosphate or fructose 2-phosphoro[35S]thioate 6-phosphate. The turnover of the thiophosphorylated enzyme intermediate as well as the overall phosphatase reaction was four times faster than with authentic fructose 2,6-bisphosphate. Fructose 2-phosphorothioate 6-phosphate was 10-100-fold less potent than authentic fructose 2,6-bisphosphate in stimulating 6-phosphofructo-1-kinase and pyrophosphate:fructose 6-phosphate phosphotransferase, but about 10 times more potent in inhibiting fructose 1,6-bisphosphatase. The analogue was twice as effective as authentic fructose 2,6-bisphosphate in stimulating pyruvate kinase from trypanosomes.     

Regulation of fructose 2,6-bisphosphate concentration in white adipose tissue.

Injection of insulin to fed rats diminished the concentration of fructose 2,6-bisphosphate in white adipose tissue. Incubation of epididymal fat-pads or adipocytes with insulin stimulated lactate release and sugar detritiation and also decreased fructose 2,6-bisphosphate concentration. Such a decrease was, however, not observed in fat-pads from starved or alloxan-diabetic rats. Incubation of adipocytes from fed rats with various concentrations of glucose or fructose led to a dose-dependent rise in fructose 2,6-bisphosphate which correlated with lactate output and detritiation of 3-3H-labelled sugar. In adipocytes from fed rats, palmitate stimulated the detritiation of [3-3H]glucose without affecting lactate production and fructose 2,6-bisphosphate concentration. Incubation of epididymal fat-pads from fed rats in the presence of antimycin stimulated lactate output but decreased fructose 2,6-bisphosphate concentration. Changes in lipolytic rates brought about by noradrenaline, insulin, adenosine and corticotropin in adipocytes from fed rats were not related to changes in fructose 2,6-bisphosphate or to rates of lactate output. In fed rats, the activity of 6-phosphofructo-2-kinase was not changed after treatment of adipocytes with insulin, noradrenaline or adenosine. It is suggested that the decrease in fructose 2,6-bisphosphate concentration observed after insulin treatment can be explained by the increase in sn-glycerol 3-phosphate, an inhibitor of 6-phosphofructo-2-kinase.     

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.     

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     

Effect of ethylene treatment on the concentration of fructose-2,6-bisphosphate and on the activity of phosphofructokinase 2/fructose-2,6-bisphosphatase in banana

Preclimacteric bananas fruits were treated for 12 h with ethylene to induce the climacteric rise in respiration. One day after the end of the hormonal treatment, the two activities of the bifunctional enzyme, phosphofructokinase 2/fructose-2,6-bisphosphatase started to increase to reach fourfold their initial value 6 days later. By contrast, the activities of the pyrophosphate-dependent and of the ATP-dependent 6-phosphofructo-1-kinases remained constant during the whole experimental period, the first one being fourfold greater than the second. The concentrations of fructose 2,6-bisphosphate and of fructose 1,6-bisphosphate increased in parallel during 4 days and then slowly decreased, the second one being always about 100-fold greater than the first. The change in fructose 2,6-bisphosphate concentration can be partly explained by the rise of the bifunctional enzyme, but also by an early increase in the concentration of fructose 6-phosphate, the substrate of all phosphofructokinases, and also by the decrease in the concentration of glycerate 3-phosphate, a potent inhibitor of phosphofructokinase 2. The burst in fructose 2,6-bisphosphate and the activity of the pyrophosphate-dependent phosphofructokinase, which is in banana the only enzyme known to be sensitive to fructose 2,6-bisphosphate, can explain the well-known increase in fructose 1,6-bisphosphate which occurs during ripening.     

Fructose 2, 6-Bisphosphate in Hypoglycemic Rat Brain

Abstract: Fructose 2,6-bisphosphate has been studied during hypoglycemia induced by insulin administration (40 IU/kg). No changes in content of cerebral fructose 2,6-bisphosphate were found in mild hypoglycemia, but the level of this compound was markedly decreased in hypoglycemic coma and recovered after 30 min of glucose administration. To correlate a possible modification of the concentration of the metabolite with selective regional damage occurring during hypoglycemic coma, we have analyzed four cerebral areas (cortex, striatum, cerebellum, and hippocampus). Fructose 2,6-bisphosphate concentrations were similar in the four areas analyzed; severe hypoglycemia decreased levels of the metabolite to the same extent in all the brain areas studied. The decrease in content of fructose 2,6-bisphosphate was not always accompanied by a parallel decrease in ATP levels, a result suggesting that the low levels of the bisphosphorylated metabolite during hypoglycemic coma could be due to the decreased 6-phosphofructo-2-kinase activity, mainly as a consequence of the fall in concentration of its substrate (fructose 6-phosphate). These results suggest that fructose 2,6-bisphosphate could play a permissive role in cerebral tissue, maintaining activation of 6-phosphofructo-l-kinase and glycolysis.     

Some properties of phosphofructokinase from kidney cortex and their relation to glucose metabolism

1. Phosphofructokinase from rat kidney cortex has been partially purified by using a combination of isoelectric and ammonium sulphate precipitation. This preparation was free of enzymes which interfered with the measurement of either product of phosphofructokinase. 2. At concentrations greater than the optimum, ATP caused inhibition which was decreased by raising the fructose 6-phosphate concentration. This suggested that ATP reduced the affinity of phosphofructokinase for the other substrate. Citrate potentiated the ATP inhibition. 3. AMP and fructose 1,6-diphosphate relieved the inhibition by ATP or citrate by increasing the affinity of the enzyme for fructose 6-phosphate. 4. K(+) is shown to stimulate and Ca(2+) to inhibit phosphofructokinase. 5. The similarity between the complex properties of phosphofructokinase from kidney cortex and other tissues (e.g. cardiac and skeletal muscle, brain and liver) suggests that the enzyme in kidney cortex tissue is normally subject to metabolic control, similar to that in other tissues.     

Yeast 6-phosphofructo-2-kinase: sequence and mutant.

We have reported yeast 6-phosphofructo-2-kinase (EC 2.7.1.105) as having a ca. 96-kDa subunit size, as well as isolation of its structural gene, PFK26. Sequencing now shows an open reading frame of 827 amino acids and 93.5 kDa. The deduced amino acid sequence has 42% identity with the 55-kDa subunit of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver with extra material at both ends. Although the yeast sequence is especially similar to the liver one in its bisphosphatase domain, the essential His-258 of the liver enzyme is, in yeast, a serine, which may explain the apparent lack of bisphosphatase activity. Also, the yeast enzyme known to be activated via protein kinase A, has a putative phosphorylation site near its C-terminus and lacks the N-terminal phosphorylation sequence involved in inhibition of the liver enzyme. In a chromosomal null mutant strain, pfk26::LEU2, activity was marginal and the protein was not detectable as antigen. The mutant strain grew well on glucose and contained a near-normal level of fructose 2,6-P2. But in its growth on pyruvate, by contrast with the wild-type strain, no fructose 2,6-P2 was detectable, and it did not form after glucose addition in the presence of cycloheximide either. Such resting cells, however, metabolized glucose at the normal high rate. Glucose addition to the pfk26 mutant strain in the absence of cycloheximide, on the other hand, caused a ca. 10% normal rate of fructose 2,6-P2 accumulation, presumably employing a glucose-inducible second enzyme. Using strains also lacking 6-phosphofructo-1-kinase, affinity chromatography revealed the second enzyme as a minor peak amounting to 6% of 6-phosphofructo-2-kinase activity in a PFK26 strain and as the sole peak, in similar amount, in a pfk26 mutant strain.     

The mechanism by which glucose increases fructose 2,6-bisphosphate concentration in Saccharomyces cerevisiae. A cyclic-AMP-dependent activation of phosphofructokinase 2

When glucose was added to a suspension of Saccharomyces cerevisiae in stationary phase, it caused a transient increase in the concentration of cyclic AMP and a more persistent increase in the concentration of hexose 6-phosphate and of fructose 2,6-bisphosphate. These effects of glucose on cyclic AMP and fructose 2,6-bisphosphate but not that on hexose 6-phosphate were greatly decreased in the presence of 0.15 mM acridine orange or when a temperature-sensitive mutant deficient in adenylate cyclase was used at the restrictive temperature. Incubation of the cells in the presence of dinitrophenol and in the absence of glucose increased the concentration of both cyclic AMP and fructose 2,6-bisphosphate, but with a minimal change in that of hexose 6-phosphate. Glucose induced also in less than 3 min a severalfold increase in the activity of 6-phosphofructo-2-kinase and this effect was counteracted by the presence of acridine orange. When a cell-free extract of yeast in the stationary phase was incubated with ATP-Mg and cyclic AMP, there was a 10-fold activation of 6-phosphofructo-2-kinase. Finally, the latter enzyme was purified 150-fold and its activity could then be increased about 10-fold upon incubation with ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase. This activation resulted from a 4.3-fold increase in V and a 2-fold decrease in Km. Both forms of the enzyme were inhibited by sn-glycerol 3-phosphate. From these results it is concluded that the effect of glucose in increasing the concentration of fructose 2,6-bisphosphate in S. cerevisiae is mediated by the successive activation of adenylate cyclase and of cyclic-AMP-dependent protein kinase and by the phosphorylation of 6-phosphofructo-2-kinase by the latter enzyme. In deep contrast with what is known of the liver enzyme, yeast 6-phosphofructo-2-kinase is activated by phosphorylation instead of being inactivated.     

Fructose 2,6-bisphosphate in yeast

Fructose 2,6-bisphosphate was identified in $\text{Saccharomyces cerevisiae}$ grown on glucose both by its property to be an acid-labile stimulator of 6-phosphofructo 1-kinase and by its ability to be quantitatively converted into fructose 6-phosphate under mild acid conditions. Fructose 2,6-bisphosphate was undetectable in cells grown on non-glucose sources. When glucose was added to the culture, fructose 2,6-bisphosphate was rapidly synthesized, reaching within 1 min concentrations able to cause a profound inhibition of fructose 1,6-bisphosphatase and a great stimulation of 6-phosphofructo 1-kinase.     

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.     

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.     

Mechanism of stimulation of endogenous fermentation in yeast by carbonyl cyanide m-chlorophenylhydrazone

Addition of the uncoupler and protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to starved yeast cells starts endogenous alcoholic fermentation lasting about 20 min. Hexose 6-phosphates, fructose 2,6-bisphosphate, and pyruvate accumulate in less than 2 min after addition of CCCP from almost zero concentration to concentrations which correspond to 1/5-1/10 of the steady-state concentrations during fermentation of glucose. CCCP immediately causes a decrease of the intracellular cytosolic pH from 6.9 to 6.4. This change activates adenylate cyclase (Purwin, C., Nicolay, K., Scheffers, W.A., and Holzer, H. (1986) J. Biol. Chem. 261, 8744-8749) and leads to the previously observed transient increase of cyclic AMP. It is shown here that the following enzymes known from in vitro experiments to be activated by cyclic AMP-dependent phosphorylation are activated in the CCCP-treated starved yeast cells in vivo: glycogen phosphorylase, trehalase (pH 7), 6-phosphofructo-2-kinase. The activation of 6-phosphofructo-2-kinase leads to an accumulation of fructose 2,6-bisphosphate, which is known from in vitro experiments to activate 6-phosphofructo-1-kinase and to inhibit fructose-1,6-bisphosphatase. All effects observed in the intact yeast cells fit with the idea that the CCCP-initiated activation of adenylate cyclase leads to a sequence of events which by protein phosphorylation and allosteric effects initiates endogenous alcoholic fermentation.     

Ribose 1,5-bisphosphate is a putative regulator of fructose 6-phosphate/fructose 1,6-bisphosphate cycle in liver

6-Phosphofructo-1-kinase and fructose-1,6-bisphosphatase are rate-limiting enzymes for glycolysis and gluconeogenesis respectively, in the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver. The effect of ribose 1,5-bisphosphate on the enzymes was investigated. Ribose 1,5-bisphosphate synergistically relieved the ATP inhibition and increased the affinity of liver 6-phosphofructo-1-kinase for fructose 6-phosphate in the presence of AMP. Ribose 1,5-bisphosphate synergistically inhibited fructose-1,6-bisphosphatase in the presence of AMP. The activating effect on 6-phosphofructo-1-kinase and the inhibitory effect on fructose-1,6-bisphosphatase suggest ribose 1,5-bisphosphate is a potent regulator of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver.     

Some Properties of Partially Purified Pyruvate Kinase from Euglena gracilis Klebs var. bacillaris

A method of purification of pyruvate kinase (EC 2.7.1.40) from light-grown Euglena gracilis var. bacillaris was developed which yielded an enzyme preparation purified 115-fold over crude extracts. During organelle formation, levels of pyruvate kinase in extracts prepared from cells engaged in light-induced chloroplast development do not change significantly. The enzyme has a molecular weight of approximately 240,000 and a requirement for both K⁺ and Mg²⁺. Fructose 1,6-diphosphate activates the enzyme when the concentration of phosphoenol-pyruvate is limiting; it does not activate when the concentration of ADP is limiting. ATP, citrate, and Ca²⁺ are inhibitors of the enzyme and inhibit the fructose 1,6-diphosphate stimulation of the enzyme activity. ATP inhibition is only partially reversed by high concentrations of fructose 1,6-diphosphate. Further reversal of inhibition can be achieved by dialysis. Ca²⁺-dependent inhibition can be reversed by a chelating agent but not by increased concentrations of Mg²⁺.     

Inhibition of fructose bisphosphatase and stimulation of phosphofructokinase by a stable isosteric phosphonate analog of fructose 2,6-bisphosphate

We describe the synthesis of a mixture of D-manno- and D-gluco-2,5-anhydro-1-deoxy-1-phosphonohexitol 6-phosphate via a Horner-Emmons reaction of 2,3,5-tri-O-benzyl-beta-D-arabinofuranose followed by phosphorylation of the equivalent 6-position and subsequent deprotection. This mixture inhibits fructose-1,6-bisphosphatase; the concentration required for half-maximal effect in the presence of 25 microM AMP is approximately 6 microM. The mixture of analogs also stimulates 6-phosphofructo-1-kinase from rabbit liver; the concentration required to reach one-half Vmax was found to be ca. 25 microM at 0.25 mM fructose 6-phosphate and 50 microM AMP. These analogs have replaced the labile anomeric phosphate of fructose 2,6-bisphosphate with a stable methylenephosphonate, and could be of great interest due to their appropriate physiological effects and their chemical stability.