Increase of 2,3-bisphosphoglycerate synthase/phosphatase during maturation of reticulocytes with high 2,3-bisphosphoglycerate content

In the rabbit and in the rat, which possess erythrocytes with high concentration of 2,3-bisphosphoglycerate, the 2,3-bisphosphoglycerate synthase activity increases more than two fold during reticulocyte maturation. Isolation of the enzymes with 2,3-bisphosphoglycerate synthase activity present in extracts of reticulocytes and mature erytrocytes by ion exchange fast liquid chromatography shows that the increase in the synthase activity is due to the accumulation of the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase (EC 2.7.5.4/EC 3.1.3.13) which represents more than 80% of the synthase activity of the cell extracts. During reticulocyte maturation phosphoglycerate mutase (EC 5.4.2.1), which makes a small contribution to the 2,3-bisphosphoglycerate synthase activity in the erythroid cells, decreases in the rabbit and remains constant in the rat.     

Modification of cys-128 of pig kidney fructose 1,6-bisphosphatase with different thiol reagents: Size dependent effect on the substrate and fructose-2,6-bisphosphate interaction

Treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide was shown to abolish the inhibition by fructose 2,6-bisphosphate, which also protected the enzyme against this chemical modification [Reyes, A., Burgos, M. E., Hubert, E., and Slebe, J. C. (1987),J. Biol. Chem. 262, 8451–8454]. On the basis of these results, it was suggested that a single reactive sulfhydryl group was essential for the inhibition. We have isolated a peptide bearing the N-ethylmaleimide target site and the modified residue has been identified as cysteine-128. We have further examined the reactivity of this group and demonstrated that when reagents with bulky groups are used to modify the protein at the reactive sulfhydryl [e.g., N-ethylmaleimide or 5,5-dithiobis-(2-nitrobenzoate)], most of the fructose 2,6-bisphosphate inhibition potential is lost. However, there is only partial or no loss of inhibition when smaller groups (e.g., cyanate or cyanide) are introduced. Kinetic and ultraviolet difference spectroscopy-binding studies show that the treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide causes a considerable reduction in the affinity of the enzyme for fructose 2,6-bisphosphate while affinity for fructose 1,6-bisphosphate does not change. We can conclude that modification of this reactive sulfhydryl affects the enzyme sensitivity to fructose 2,6-bisphosphate inhibition by sterically interfering with the binding of this sugar bisphosphate, although this residue does not seem to be essential for the inhibition to occur. The results also suggest that fructose 1,6-bisphosphate and fructose 2,6-bisphosphate may interact with the enzyme in a different way.     

The interaction of fructose 2,6-bisphosphate with an allosteric site of rat liver fructose 1,6-bisphosphatase

Rat liver fructose 1,6-bisphosphatase can be protected against partial inactivation by N-ethylmaleimide by low concentrations of fructose 2,6-bisphosphate or high concentrations of fructose 1,6-bisphosphate. The partially inactivated enzyme has a much reduced sensitivity to high substrate inhibition and has lost the sigmoid component of the inhibition by fructose 2,6-bisphosphate; this compound is a simple linear competitive inhibitor of the modified enzyme. The results suggest that fructose 2,6-bisphosphate can bind to the enzyme at two distinct sites, the catalytic site and an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit by binding to the allosteric site.     

Biphasic effect of fructose 2,6-bisphosphate on the liver fructose-1,6-bisphosphatase: mechanistic and physiological implications

Fructose 2,6-bisphosphate has been claimed to be both a substrate analogue and an allosteric inhibitor of fructose-1,6-bisphosphatase. The results reported here show that fructose 2,6-bisphosphate can be both an inhibitor and an activator of the enzyme, depending on the substrate concentration. This biphasic behaviour at saturating concentrations of substrate can only be due to an allosteric effect. In addition to the mechanistic implication it is possible that this finding may have physiological meaning.     

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate levels in erythrocytes with high and low 2,3-bisphosphoglycerate content during postnatal development

In rabbit and sheep erythrocytes the concentrations of 2,3-bisphosphoglycerate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate suffer important changes after birth, which differ in both species. The changes of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate correlate with the changes in the levels of the enzymatic activities involved in their synthesis. The change of 2,3-bisphosphoglycerate levels in rabbit but not in sheep erythrocytes could be explained by the changes of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios.     

Hormonal control of fructose 2,6-bisphosphate concentration and of phosphofructokinase 2 in the rat liver during development

In fetal rat liver the concentration of fructose 2,6-bisphosphate is decreased by administration of glucagon. The glucagon effect, i.e., the phosphorylation state of phosphofructokinase 2, dominates over the substrate supply. Insulin was found to increase fructose 2,6-bisphosphate only when exogenous glucose is supplied simultaneously. The total activity of phosphofructokinase 2 exhibits remarkable developmental changes. It is high at term, moderate in the fetal as well as in the mature organ, and low during suckling. The level of the enzyme during development is controlled by pancreatic and adrenal hormones.     

Crystal structure of human muscle aldolase complexed with fructose 1,6-bisphosphate: mechanistic implications

Fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the epsilon-amino group of Lys229. The existing apo-enzyme structure was refined using the crystallographic free-R-factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6-bisphosphate), and the crystal structure of this complex has been determined to 2.8 A. The apo structure differs from the previous Brookhaven-deposited structure (1ald) in the flexible C-terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen-bonded Michaelis complex before the formation of the covalent complex.     

Fructose 2,6-bisphosphate as a contaminant of commercially obtained fructose 6-phosphate: Effect on PPi:fructose 6-phosphate phosphotransferase

Fructose 6-phosphate from several commercial sources was shown to be contaminated with fructose 2,6-bisphosphate. This contaminant was identified by its activation of PP_i:fructose 6-phosphate phosphotransferase, extreme acid lability and behaviour on ion-exchange chromatography. The apparent kinetic properties of PP_i:fructose 6-phosphate phosphotransferase from castor bean endosperm were considerably altered when contaminated fructose 6-phosphate was used as a substrate. Varying levels of fructose 2,6-bisphosphate in the substrate may account for differences that have been observed in the properties of the above enzyme from several plant sources.     

The effect of fructose 2,6-bisphosphate and AMP on the activity of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase from rat liver

Rat liver fructose-1,6-bisphosphatase was partially phosphorylated in vitro and separated into unphosphorylated and fully phosphorylated enzyme. The effects of fructose 2,6-bisphosphate and AMP on these two enzyme forms were examined. Unphosphorylated fructose-1,6-bisphosphatase was more easily inhibited by both effectors. Fructose 2,6-bisphosphate affected both K0.5 and Vmax, while the main effect of AMP was to lower Vmax. Fructose 2,6-bisphosphate and AMP together acted synergistically to decrease the activity of fructose-1,6-bisphosphatase, and since unphosphorylated and phosphorylated enzyme forms are affected differently, this might be a way to amplify the effect of phosphorylation.     

Identification of a nerve ending-enriched 29-kDa protein,labeled with [3-32P]1,3-bisphosphoglycerate,as monophosphoglycerate mutase: inhibition by fructose-2,6-bisphosphate via enhancement of dephosphorylation

Glucose metabolism is of vital importance in normal brain function. Evidence indicates that glycolysis, in addition to production of ATP, plays an important role in maintaining normal synaptic function. In an effort to understand the potential involvement of a glycolytic intermediate(s) in synaptic function, we have prepared [3-32P]1,3-bisphosphoglycerate and [32P]3-phosphoglycerate and sought their interaction with a specific nerve-ending protein. We have found that a 29-kDa protein is the major component labeled with either [3-32P]1,3-bisphosphoglycerate or [32P]3-phosphoglycerate. The protein was identified as monophosphoglycerate mutase (PGAM). This labeling was remarkably high in the brain and synaptosomal cytosol fraction, consistent with the importance of glycolysis in synaptic function. Of interest, fructose-2,6-bisphosphate (Fru-2,6-P2) inhibited PGAM phosphorylation and enzyme activity. Moreover, Fru-2,6-P2 potently stimulated release of [32P]phosphate from the 32P-labeled PGAM (EC50 = 1 microM), suggesting that apparent reduction of PGAM phosphorylation and enzyme activity by Fru-2,6-P2 may be due to stimulation of dephosphorylation of PGAM. The significance of these findings is discussed.     

Metabolic integration in locust flight: the effect of octopamine on fructose 2,6-bisphosphate content of flight muscle in vivo

The biogenic amine octopamine was injected into the haemolymph of 20-days old male locusts,Locusta migratoria, and the content of fructose 2,6-bisphosphate, a potent activator of glycolysis, was measured in the flight muscle after various time. Octopamine brought about a transient increase in fructose 2,6-bisphosphate. After the injection of 10 l of 10 mmol·l^-1 d, l-octopamine fructose 2,6-bisphosphate was increased by 61% within 2 min. Ten minutes after the injection fructose 2,6-bisphosphate was increased to 6.71±0.89 nmol·g^-1 flight muscle, almost 300% over the control value. Flight caused fructose 2,6-bisphosphate in flight muscle to decrease, but this decrease was counteracted by octopamine injected into the haemolymph of flying locusts. Octopamine and fructose 2,6-bisphosphate may act as signals to stimulate the oxidation of carbohydrate and to integrate muscle performance and metabolism. This mechanism appears particularly significant in the initial stage of flight when carbohydrates are the main fuel.Abbreviations F2,6P₂ fructose 2,6-bisphosphate - F6P fructose 6-phosphate - PFK1 6-phosphofructokinase (EC 2.7.1.11) - P _i inorganic phosphate - PP _i -PFK pyrophosphate dependent fructose 6-phosphate phosphotransferase (EC 2.7.1.90)     

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

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

Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: Binding of substrate induces a second class of site with lowered affinity and catalytic activity

Background

Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P₂ and by high concentrations of its substrate Fru-1,6-P₂. The mechanism that produces substrate inhibition continues to be obscure.

Methods

Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P₂ and Fru-1,6-P₂ on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244.

Results

The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P₂ induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P₂, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a “stapler” that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition.

Conclusions

Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits.

General significance

Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.     

Carbon partitioning in Norway spruce: amounts of fructose 2,6-bisphosphate and of intermediates of starch/sucrose synthesis in relation to needle age and degree of needle loss

Summary Intermediates involved in carbon partitioning between starch and sucrose [dihydroxyacetone phosphate + glyceraldehyde 3-phosphate (TP), 3-phosphoglyceric acid, fructose 6-phosphate (F6P), fructose 2,6-bisphosphate (F26BP), in addition to glucose, fructose, sucrose and starch] were analysed in lyophilized needles of Norway spruce (Picea abies L. Karst). Samples were taken from all distinct parts of first and second order branches and the analysed data related to season, needle age, needle position and degree of needle loss (control and class 2 approx. 30%–40% needle loss). Positive and inverse correlations of F26BP, an important regulator of carbon partitioning between starch and sucrose, and F6P or TP existed in all samples. F26BP levels were highest in developing needles and gradually decreased during maturation, which is possibly indicative of changes in the relative sink strength during development (switch from import to export of sucrose). In class 2 needles the amount of F26BP was significantly increased. Together with nearly unaltered levels of sucrose but only slightly decreased amounts of starch the results can be taken as evidence for impaired carbon export in our class 2 samples. The data are discussed with respect to needle development and a possible impact of both air pollutants and mineral deficiency at the location from which the samples were taken.     

Cloning and molecular characterization of fructose-1,6-bisphosphate aldolase gene regulated by high-salinity and drought in Sesuvium portulacastrum

Sesuvium portulacastrum, a mangrove plant from seashore, is a halophyte species well adapted to salinity and drought. Some efforts have been made to describe its physiological and structural characteristics on salt and drought-tolerance, but the underlying molecular mechanism and key components have not yet been identified. Here, a fructose-1,6-bisphosphate aldolase gene, designated SpFBA, was isolated and characterized from S. portulacastrum roots in response to seawater. The SpFBA cDNA has a total length of 1452 bp with an open reading frame of 1071 bp, and is predicted to encode a precursor protein of 357 amino acid residues sharing high degree of homology with class I FBAs from other plants. Semi-quantitative RT-PCR analysis indicated that the SpFBA was more strongly expressed in roots than in leaves and stems, and the abiotic stimuli such as Seawater, NaCl, ABA, and PEG, could trigger a significant induction of SpFBA in S. portulacastrum roots within 2–12 h. Overproduction of Recombinant SpFBA resulted in an increased tolerance to salinity in transgenic Escherichia coli. All these results suggest that the SpFBA plays very important roles in responding to salt stress and related abiotic stimuli, and in improving the survival ability of S. portulacastrum under high salinity and drought. The GenBank Accession number of S. portulacastrum fructose-1,6-bisphosphate aldolase (SpFBA) is ACG68894.     

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

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

Changes in the concentration of cAMP, fructose 2,6-bisphosphate and related metabolites and enzymes in Saccharomyces cerevisiae during growth on glucose

Changes in the concentration of several metabolites and enzymes related to carbohydrate metabolism were measured during the growth of Saccharomyces cerevisiae on a mineral medium containing glucose as the limiting nutrient. When about 50% of the original glucose was used the exponential phase ended and the culture entered a 'transition' phase before the complete exhaustion of glucose. In this transition phase several metabolic changes occurred. cAMP, that decreased along growth, reached a constant value of about 0.7 nmol/g dry weight. A pronounced drop in fructose-6-phosphate-2-kinase activity and in the concentration of fructose 2,6-bisphosphate and fructose 1,6-bisphosphate was observed accompanied by a less marked decrease in hexose monophosphates. Trehalase activity also dropped and reached a minimal value at the onset of the stationary phase when synthesis of trehalose began. Glycogen concentration and glycogen synthase activity increased sharply during the transition phase. Plasma membrane ATPase began to increase at the middle of the exponential phase and then, coincident with the glucose exhaustion, a 90% decrease in the measurable activity was observed.     

Immuno-gold localization of cytochrome f,light-harvesting complex,ATP synthase and ribulose 1,5-bisphosphate carboxylase/oxygenase

The proteins ribulose 1,5-bisphosphate carboxylase/oxygenase, ATP synthase, light-harvesting chlorophyll a/b protein, and cytochrome f, have been localized in mesophyll chloroplasts of barley (Hordeum vulgare L.) by electron microscopy of immunogold-labelled sections. The light-harvesting chlorophyll a/b protein and cytochrome f are shown to be present in the grana, both within the stacks and at the margins, and in the stromal membranes. Although the absolute amount of labelling for these proteins is greater in the grana than in the stromal membranes, when expressed as label/membrane length the partitioning appears approximately equal between appressed and non-appressed membranes for both the light-harvesting chlorophyll a/b protein and cytochrome f. ATP synthase is restricted to the non-appressed thylakoid membranes, and ribulose 1,5-bisphosphate carboxylase/oxygenase is uniformly distributed through the stromal contents.Abbreviations CF₁ ATP synthase - LHCPII light-harvesting chlorophyll a/b protein - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase