Fructose 2,6-bisphosphate and plant carbohydrate metabolism

The control of the fructose 2,6-bisphosphate (Fru2,6P₂) concentration and its possible role in controlling carbohydrate synthesis and degradation are discussed. This regulator metabolite is involved in the fine tuning of photosynthetic metabolism, and in controlling photosynthetic partitioning, and may also be involved in the response to hormones, wounding, and changing water relations. Study of the mechanisms controlling Fru2,6P₂ concentrations could reveal insights into how these responses are mediated. However, the detailed action of Fru2,6P₂ requires more attention, especially in respiratory metabolism where the background information about the compartmentation of metabolism between the plastid and cytosol is still inadequate, and the potential role of pyrophosphate has to be clarified.     

Fructose 2,6-bisphosphate, a potent regulator of carbohydrate metabolism, inhibits trehalose phosphorylase from protist Euglena gracilis

Partially purified trehalose phosphorylase (EC 2.4.1.64) from Euglena gracilis SM-ZK was inhibited by fructose 2,6-bisphosphate in both synthetic and degradative directions. Ki value for trehalose phosphorolysis was 1.2 microM and that for trehalose synthesis was 0.5 microM. Functions of fructose 2,6-bisphosphate in Euglena, particularly in the regulative mechanism of the two reserve carbohydrates, paramylon and trehalose, are discussed.     

Fructose 2,6-bisphosphate and carbohydrate metabolism during the life cycle of the aquatic fungus Blastocladiella emersonii

Removal of the growth medium and resuspension of Blastocladiella emersonii vegetative cells in a sporulation medium resulted in an abrupt fall of fructose 2,6-bisphosphate concentration to about 2% of its initial value within 10 min. The concentrations of hexose 6-phosphate and of fructose 1,6-bisphosphate also decreased by, respectively, three and tenfold over the same period. All these values remained at their low level throughout the sporulation phase and during the subsequent germination of zoospores when performed in the absence of glucose. In contrast, the concentration of cyclic AMP was low during the sporulation period and exhibited a transient increase a few minutes after the initiation of germination. Other biochemical events occurring during sporulation were a 70% reduction in glycogen content and the complete disappearance of trehalose. The remaining glycogen was degraded upon subsequent germination of the zoospores. B. emersonii phosphofructo 2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) could not be separated from each other by various chromatographic procedures, suggesting that they were part of a single bifunctional protein. On anion-exchange chromatography, two peaks of PFK-2 and FBPase-2 were resolved. Upon incubation of fractions from the two peaks or of a crude extract in the presence of [2-32P]fructose 2,6-bisphosphate, two radiolabelled subunits with molecular masses close to 90 and 54 kDa were obtained. The labelling of the subunit of higher molecular mass was greater than that of the lower one in extracts prepared in the presence of protease inhibitors and in the first peak of the Mono Q column. PFK-2 and FBPase-2 displayed kinetic properties comparable to those of mammalian enzymes, but no indication of a cyclic AMP-dependent regulation could be obtained. Phosphofructo 1-kinase and fructose-1,6-bisphosphatase from B. emersonii were, respectively, stimulated and inhibited by micromolar concentrations of fructose 2,6-bisphosphate. The physiological significance of these properties is discussed. A simple method for the determination of trehalose is also reported.     

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.     

Fructose 2,6-bisphosphate in human erythrocytes

The fructose 2,6-bisphosphate concentrations in unwashed, washed, and leukocyte-free erythrocytes were compared. The concentration in washed red cells was 31 +/- 15 pmol per ml of cells (mean +/- S.D., n = 6). The concentration in unwashed erythrocytes was at least twofold higher, but the value in washed red cells was not due to leukocyte contamination because it did not decrease further when washed cells were passed through an Imgard column, which would have removed any remaining leukocytes. No platelets were detected among the washed erythrocytes. Thus, the concentration in erythrocytes after washing was ascribed solely to these cells. The fructose 2,6-bisphosphate concentration did not change when the glycolytic activity varied with pH, indicating that this compound is not involved in the regulation of carbohydrate metabolism in erythrocytes under these conditions.     

Fructose 2,6-bisphosphate inhibition of phosphoglucomutase

Fructose 2,6-bisphosphate inhibits phosphoglucomutase noncompetitively with respect to the cofactor glucose 1,6-bisphosphate. Previous studies from our laboratory had shown that phosphoglucomutase was activated by fructose 2,6-bisphosphate in the absence of added glucose 1,6-bisphosphate. The fructose 2,6-bisphosphate activation previously reported was due to the presence of glucose 1,6-bisphosphate in the commercial preparation of fructose 2,6-bisphosphate.     

Fructose 2,6-bisphosphate in isolated foetal hepatocytes

Fru 2,6-P2 was present in isolated foetal hepatocytes at a concentration of 1.6 nmol per g cells. When foetal hepatocytes were exposed to glucagon no changes were observed either in the concentration of Fru 2,6-P2 and lactate release or in the activities of 6-phosphofructo-2-kinase and pyruvate kinase. Incubation of purified 6-phosphofructo-2-kinase with the catalytic subunit of protein kinase did not change the enzyme activity. The inhibition by sn-glycerol 3-phosphate was much lower for the foetal than for adult enzyme. These results suggest that an isoenzyme of 6-phosphofructo-2-kinase in foetal hepatocytes different from that of adult hepatocytes may be present.     

Fructose 2,6-bisphosphate and the climacteric in bananas

This work was done to test the view that there is a marked rise in the content of fructose 2,6-bisphosphate during the climacteric of the fruit of banana (Musa cavendishii Lamb ex. Paxton). Bananas were ripened in the dark in a continuous stream of air in the absence of exogenous ethylene. CO2 production and the contents of fructose 2,6-bisphosphate and sucrose were monitored over a 15-day period. A range of extraction procedures for fructose 2,6-bisphosphate were compared. Recovery of fructose 2,6-bisphosphate added to samples of unripe fruit varied from poor to unmeasurable. Recoveries from samples of ripe fruit were high. It is argued that this differential recovery of fructose 2,6-bisphosphate undermines claims that the amount of this compound increases at the climacteric. When recoveries are taken into account, our data suggest that there is no major change in fructose 2,6-bisphosphate content during the onset of the climacteric in bananas.     

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate in erythrocytes during chicken development

In contrast to mammalian erythrocytes, chicken erythrocytes contain fructose 2,6-bisphosphate at levels (0.5 nmol/10(9) cells) similar to those of 2,3-bisphosphoglycerate (1.2 nmol/10(9) cells) and slightly lower than those of glucose 1,6-bisphosphate (5.2 nmol/10(9) cells). In chick embryo erythrocytes the levels of both fructose 2,6-bisphosphate and glucose 1,6-bisphosphate are much lower. They begin to increase at hatching and reach the levels in chicken in a few days.     

Fructose 2,6-bisphosphate hydrolyzing enzymes in higher plants

The phosphatases that hydrolyze fructose 2,6-bisphosphate in a crude spinach (Spinacia oleracea L.) leaf extract were separated by chromatography on blue Sepharose, into three fractions, referred to as phosphatases I, II, and III, which were further purified by various means. Phosphatase I hydrolyzed fructose 2,6-bisphosphate, with a K_m value of 30 micromolar, to a mixture of fructose 2-phosphate (90%) and fructose 6-phosphate (10%). It acted on a wide range of substrates and had a maximal activity at acidic pH. Phosphatase II specifically recognized the osyl-link of phosphoric derivatives and had more affinity for the β-anomeric form. Its apparent K_m for fructose 2,6-bisphosphate was 30 micromolar. It most likely corresponded to the fructose-2,6-bisphosphatase described by F. D. Macdonald, Q. Chou, and B. B. Buchanan ([1987] Plant Physiol 85: 13-16). Phosphatase III copurified with phosphofructokinase 2 and corresponded to the specific, low-K_m (24 nanomolar) fructose-2,6-bisphosphatase purified and characterized by Y. Larondelle, E. Mertens, E. Van Schaftingen, and H. G. Hers ([1986] Eur J Biochem 161: 351-357). Three similar types of phosphatases were present in a crude extract of Jerusalem artichoke (Helianthus tuberosus) tuber. The concentration of fructose 2,6-bisphosphate decreased at a maximal rate of 30 picomoles per minute and per gram of fresh tissue in slices of Jerusalem artichoke tuber, upon incubation in 50 millimolar mannose. This rate could be accounted for by the maximal extractable activity of the low-K_m fructose-2,6-bisphosphatase. A new enzymic method for the synthesis of β-glucose 1,6-bisphosphate from β-glucose 1-phosphate and ATP is described.     

Fructose 2,6-bisphosphate in rat erythrocytes. Inhibition of fructose 2,6-bisphosphate synthesis and measurement by glycerate 2,3-bisphosphate.

The concentration of fructose 2,6-bisphosphate found in freshly isolated erythrocytes was below the limit of detection (20 pmol/ml of packed cells). However, it increased to about 250 pmol/ml of cells when erythrocytes were incubated with glucose at pH 6.9, but not at pH 7.4 or 8.2. This could be explained by variations in the content of glycerate 2,3-bisphosphate, which was found to inhibit 6-phosphofructo-2-kinase, the enzyme responsible for fructose 2,6-bisphosphate synthesis. Glycerate 2,3-bisphosphate was also found to inhibit the potato enzyme (pyrophosphate:fructose-6-phosphate 1-phosphotransferase) used for the measurement of fructose 2,6-bisphosphate.     

Stimulation of yeast phosphofructokinase activity by Fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate is a powerful activator of yeast phosphofructokinase when assayed at pH levels of ≥7.0. Half maximal stimulation of enzyme activity occurs at 10^?7 M levels of Fru 2,6-P₂ concentration. This stimulating effect by Fru 2,6-P₂ can be synergistic to that exerted by AMP in counteracting the inhibition of phosphofructokinase activity by ATP. The affinity (S_0.5) of the yeast enzyme to fructose 6-phosphate changes from 1.5 mM in the absence of Fru 2,6-P₂ to 40 μM in its presence.     

Fructose 2,6-bisphosphate and glycolytic oscillations in skeletal muscle extracts

Oscillatory behavior of glycolysis in cell-free extracts of rat skeletal muscle involves bursts of phosphofructokinase activity due to autocatalytic activation by fructose-1,6-P2. Fructose-2,6-P2 is an even more potent activator of phosphofructokinase and is competitive with fructose-1,6-P2 in binding and kinetic studies. The possible role and effects of fructose-2,6-P2 on the oscillating system were therefore examined. When muscle extracts were provided with 1 mM ATP and 10 mM glucose, fructose-2,6-P2 slowly accumulated to 50 nM in 1 h. The nearly monotonic rise, in contrast to the 50-fold oscillations in fructose-1,6-P2, indicated no involvement of fructose-2,6-P2 in the oscillatory process. Addition of 0.5 microM fructose-2,6-P2 blocked the oscillations, and there was negligible appearance of glycolytic intermediates from fructose-1,6-P2 to phosphoenolpyruvate, although similar amounts of lactate accumulated. In the presence of 0.2 microM fructose-2,6-P2, there were small, transient accumulations of fructose-1,6-P2, suggesting aborted activations of phosphofructokinase. Oscillations were not blocked by 0.1 microM fructose-2,6-P2. The average [ATP]/[ADP] ratio in the presence of 0.2 or 0.5 microM fructose-2,6-P2 was half the value in its absence, demonstrating the advantage of the oscillatory behavior in maintaining a high energy state. In the presence of higher, near physiological levels of ATP and citrate, inhibitors which reduce the affinity of phosphofructokinase for fructose-2,6-P2, glycolytic oscillations were not blocked by 1 microM fructose-2,6-P2, its approximate concentration in vivo.     

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

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