The rate of substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate in skeletal muscle from cold-exposed, hyperthyroid or acutely exercised rats.

The effects of cold-exposure, the hyperthyroid state and a single exercise bout in vivo on the maximal enzyme activities of 6-phosphofructokinase and fructose-1,6-bisphosphatase in vastus lateralis muscle and the rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling measured in epitrochlearis muscle in vitro were investigated. In all cases significant changes in substrate cycling rates were observed, whether in the absence of added hormones in vitro (acute exercise), or when stimulated by insulin plus adrenaline (cold-exposure), or with respect to the catecholamine-sensitivity of the cycling rate (the hyperthyroid state).     

A hysteretic cycle in glucose 6-phosphate metabolism observed in a cell-free yeast extract

The dynamics of a partial glycolytic reaction sequence which converts glucose 6-phosphate to triose phosphates is described. The study was performed with cell-free extracts from baker's yeast harvested in the logarithmic and stationary growth phases. The experiments are based on a flow-through reactor supplied with the desalted cell-free extract as well as glucose 6-phosphate, ATP and phosphoenolpyruvate. In the reaction system the quasi-irreversible reactions catalyzed by 6-phosphofructo-1-kinase, pyruvate kinase, and fructose-1,6-bisphosphatase are involved. When substrate is supplied continuously, only stable stationary states can be observed. With transient perturbations of the substrate supply, multiple stationary states appear. Cyclic transitions between unique stable stationary states were induced by appropriate changes of the rate of substrate supply. A hysteretic cycle could then be demonstrated when, during reverse transitions, a parameter region of multistability was passed. The presence (in resting yeast) or absence (in growing yeast) of fructose-1,6-bisphosphatase did not significantly influence the dynamic capabilities of the investigated reaction sequence. The kinetic properties of the cell-free extracts fit mathematical models developed for in vitro systems reconstituted from purified enzymes.     

The effect of fructose 2,6-bisphosphate on muscle fructose-1,6-bisphosphatase activity

Rat and rabbit muscle fructose 1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) are inhibited by fructose 2,6-bisphosphate. In contrast with the liver isozyme, the inhibition of muscle fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate is not synergistic with that of AMP. Activation of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate has been observed at high concentrations of substrate. An attempt is made to correlate changes in concentrations of hexose monophosphate, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate with changes in fluxes through 6-phosphofructokinase and fructose-1,6-bisphosphatase in isolated epitrochlearis muscle challenged with insulin and adrenaline.     

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: I. Regulation of Carbon Metabolism and Succinate as a Fermentation Product

The onset of anaerobiosis in darkened, N-limited cells of the green alga Selenastrum minutum (Naeg.) Collins elicited the following metabolic responses. There was a rapid decrease in energy charge from 0.85 to a stable lower value of 0.6 accompanied by rapid increases in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also a large increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase, can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase activity. These changes resulted in an approximately twofold increase in the rate of starch breakdown indicating a Pasteur effect. The Pasteur effect was accompanied by accumulation of d-lactate, ethanol and succinate as fermentation end-products, but not malate. Accumulation of succinate was facilitated by reductive carbon metabolism by a partial TCA cycle (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). An initial stoichiometric decline in aspartate and increases in succinate and alanine suggests that aspartate catabolism provides an initial source of carbon for reduction to succinate under anoxic conditions. These observations allow us to develop a model for the regulation of anaerobic carbon metabolism and a model for short-term and long-term strategies for succinate accumulation in a green alga.     

Mathematical model of stabilization of circadian rhythm in cell energy metabolism

A mathematical model for circadian self-oscillation in the carbohydrate branch of energy metabolism (CEM) was analysed. The self-oscillations are due to the reciprocal regulation of the activities of 6-phosphofructokinase and fructose-1,6-bisphosphatase by fructose-1,6-bisphosphate. The circadian period was shown to be insensitive to metabolic disturbances because of the presence in CEM of negative feedback mechanisms regulating the activities of the key enzymes 6-phosphofructokinase, fructose-1,6-bisphosphatase, pyruvate kinase and phosphoenolpyruvate carboxykinase. It has been also shown that such mechanisms are largely synergistic in their action.     

Studies on rapid reversible and non-reversible inactivation of fructose-1,6-bisphosphatase and malate dehydrogenase in wild-type and glycolytic block mutants of Saccharomyces cerevisiae

Experimental conditions have been elaborated to test for reversibility of the malate dehydrogenase inactivation (E.C.1.1.1.37) after addition of glucose to derepressed yeast cells. Malate dehydrogenase inactivation was shown to be irreversible at all stages of inactivation. In contrast fructose-1,6-bisphosphatase inactivation (E.C.3.1.11) remained reversible for at least 30 min after addition of glucose. Rapid reversible inactivation of fructose-1,6-bisphosphatase and irreversible inactivation of malate dehydrogenase were additionally investigated in glycolytic block mutants. Normal inactivation kinetics were observed in mutants without catalytic activity of phosphoglucose isomerase (E.C.5.3.1.9), phosphofructokinase (E.C.2.7.1.11), triosephosphate isomerase (E.C.5.3.1.1) and phosphoglycerate kinase (E.C.2.7.2.3). Hence, neither type of inactivation depended on the accumulation of any glucose metabolite beyond glucose-6-phosphate. Under anaerobic conditions irreversible inactivation was completely abolished in glycolytic block mutants. In contrast rapid reversible inactivation was independent of energy provided by respiration or fermentation. Reversibility of fructose-1,6-bisphosphatase inactivation was tested under conditions which prevented irreversible malate dehydrogenase inactivation. In these experiments, fructose-1,6-bisphosphatase inactivation remained reversible for at least 120 min, whereas reversibility was normally restricted to about 30 min. This indicated a common mechanism between the irreversible part of fructose-1,6-bisphosphatase inactivation and irreversible malate dehydrogenase inactivation.     

Vanadate inhibits fructose-2,6-bisphosphatase and leads to an inhibition of sucrose synthesis in barley leaves

Vanadate (0.1–1 mM) was supplied to leaves of barley (Hordeum vulgare var. Roland) via the transpiration stream. It led to a selective inhibition of the rate of photosynthesis at high light without altering the initial slope of the light response curve, produced markedly biphasic photosynthesis induction kinetics, and selectively decreased sucrose synthesis compared to starch synthesis. There was a 3-fold increase of the steady state level of the signal metabolite fructose-2,6-bisphosphate in near saturating light. Fructose-2,6-bisphosphate is a potent inhibitor of cytosolic fruc-tose-l,6-bisphosphatase and, in agreement, the fructose-1,6-bisphosphatc level doubled. The increase of fructose-2,6-bisphosphate could not be accounted for by the known regulation of fructose-6-phosphate,2-kinase and fructose 2,6-bisphosphatase by 3-phosphoglycerate and fiuctose-6-phosphate, because these metabolites remained constant or even changed in the opposite direction to that required to generate an increase of fructose-2,6-bisphosphate. Instead, vanadate strongly inhibited the hydrolysis of fructose-2,6-bisphosphate in extracts, producing a half maximal inhibition at 2 \nM and 50 \iM in assays designed to preferentially measure the high-and low-affinity forms of fructose-2,6-bisphosphatase, respectively. Vanadale had no effect on fructosc-6-phosphate,2-kinase activity at these concentrations. Vanadate also led to a deactivation of sucrose phosphate synthase. The results are discussed in relation to the role of fructose-2,6-bisphosphate in regulating sucrose synthesis, and its interaction with the 'coarse' control of sucrose phosphate synthase.     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Liver metabolism in cold hypoxia: a comparison of energy metabolism and glycolysis in cold-sensitive and cold-resistant mammals

The effects of cold hypoxia were examined during a time-course at 2 °C on levels of glycolytic metabolites: glycogen, glucose, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, pyruvate, lactate and energetics (ATP, ADP, AMP) of livers from rats and columbian ground squirrels. Responses of adenylate pools reflected the energy imbalance created during cold hypoxia in both rat and ground squirrel liver within minutes of organ isolation. In rat, ATP levels and energy charge values for freshly isolated livers were 2.54 mol·g^-1 and 0.70, respectively. Within 5 min of cold hypoxia, ATP levels had dropped well below control values and by 8 h storage, ATP, AMP, and energy charge values were 0.21 mol·g^-1, 2.01 mol·g^-1, and 0.17, respectively. In columbian ground squirrels the patterns of rapid ATP depletion and AMP accumulation were similar to those found in rat. In rat liver, enzymatic regulatory control of glycolysis appeared to be extremely sensitive to the decline in cellular energy levels. After 8 h cold hypoxia levels of fructose-6-phosphate decreased and fructose-1,6-bisphosphate increased, thus reflecting an activation of glycolysis at the regulatory step catalysed by phospho-fructokinase fructose-1,6-bisphosphatase. Despite an initial increase in flux through glycolysis over the first 2 min (lactate levels increased 3.7 mol·g^-1), further flux through the pathway was not permitted even though glycolysis was activated at the phosphofructokinase/fructose-1,6-bisphosphatase locus at 8 h, since supplies of phosphorylated substrate glucose-1-phosphate or glucose-6-phosphate remained low throughout the duration of the 24-h period. Conversely, livers of Columbian ground squirrels exhibited no activation or inactivation of two key glycolytic regulatory loci, phosphofructokinase/fructose-1,6-bisphosphatase and pyruvate kinase/phosphoenolpyruvate carboxykinase and pyruvate carboxylase. Although previous studies have shown similar allosteric sensitivities to adenylates to rat liver phospho-fructokinase, there was no evidence of an activation of the pathway as a result of decreasing high energy adenylate, ATP or increasing AMP levels. The lack of any apparent regulatory control of glycosis during cold hypoxia may be related to hibernator-specific metabolic adaptations that are key to the survival of hypothermia during natural bouts of hibernation.Abbreviations DHAP dihydroxyacetonephosphate - EC energy charge - F1,6P₂ fructose-1,6-bisphosphate - F2,6P₂ fructose-2,6-bisphosphate - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphatase - G1P glucose-1-phosphate - G6P glucose-6-phosphate - GAP glyceraldehyde-3-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - L/R lactobionate/raffinose-based solution - MR metabolic rate - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PEPCK & PC phosphoenolpyruvate carboxykinase and pyruvate carboxylase - PFK phosphofructokinase; PK, pyruvate kinase - Q ₁₀ the effect of a 10 °C drop in temperature on reaction rates (generally, Q ₁₀=2–3) - TA total adenylates - UW solution University of Wisconsin solution (L/R-based)     

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.     

HETEROLOGOUS EXPRESSION OF Escherichia coli FRUCTOSE-1,6-BISPHOSPHATASE IN Corynebacterium glutamicum AND EVALUATING THE EFFECT ON CELL GROWTH AND L-LYSINE PRODUCTION

Fructose-1,6-bisphosphatase (FBPase), which is mainly used to supply NADPH, has an important role in increasing L-lysine production by Corynebacterium glutamicum. However, C. glutamicum FBPase is negatively regulated at the metabolic level. Strains that overexpressed Escherichia coli fructose-1,6-bisphosphatase in C. glutamicum were constructed, and the effects of heterologous FBPase on cell growth and L-lysine production during growth on glucose, fructose, and sucrose were evaluated. The heterologous fructose-1,6-bisphosphatase is insensitive to fructose 1-phosphate and fructose 2,6-bisphosphate, whereas the homologous fructose-1,6-bisphosphatase is inhibited by fructose 1-phosphate and fructose 2,6-bisphosphate. The relative enzyme activity of heterologous fructose-1,6-bisphosphatase is 90.8% and 89.1% during supplement with 3 mM fructose 1-phosphate and fructose 2,6-bisphosphate, respectively. Phosphoenolpyruvate is an activator of heterologous fructose-1,6-bisphosphatase, whereas the homologous fructose-1,6-bisphosphatase is very sensitive to phosphoenolpyruvate. Overexpression of the heterologous fbp in wild-type C. glutamicum has no effect on L-lysine production, but fructose-1,6-bisphosphatase activities are increased 9- to 13-fold. Overexpression of the heterologous fructose-1,6-bisphosphatase increases L-lysine production in C. glutamicum lysC ^T311I by 57.3% on fructose, 48.7% on sucrose, and 43% on glucose. The dry cell weight (DCW) and maximal specific growth rate (μ) are increased by overexpression of heterologous fbp. A “funnel-cask” diagram is first proposed to explain the synergy between precursors supply and NADPH supply. These results lay a definite theoretical foundation for breeding high L-lysine producers via molecular target.     

Modulation of Chloroplast Fructose-1,6-bisphosphatase Activity by Light

Inhibitor experiments indicate that light effect mediator_II which is reductively activated by transfer of electrons from the photosynthetic electron transport system at or beyond ferredoxin, is involved in activation by light of fructose-1,6-bisphosphatase in the pea plant. Activation proceeds optimally when the pH is low and Mg²⁺ is 10 millimolar. Modulation by light results in increases in maximal velocity, apparently as a result of changes in enzyme conformation. Pea leaf thylakoids are effective in modulating the activity of glyceraldehyde-3-phosphate dehydrogenase but not of fructose-1,6-bisphosphatase or glucose-6-phosphate dehydrogenase in Kalanchoë stromal extracts. There is apparently species specificity for modulation of some, but not all, of the modulatable enzymes.     

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: II. Assimilation of Ammonium by Anaerobic Cells

The green alga Selenastrum minutum (Naeg.) Collins is able to assimilate NH₄⁺ in the dark under anaerobic conditions (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). In the present study, analysis of metabolites following addition of NH₄⁺ to cells acclimated to anaerobic conditions has shown the following. There was a transient decline in adenylate energy charge from 0.6 to 0.4 followed by a recovery back to ~0.6. This was accompanied by a rapid increase in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also an increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase. These changes resulted in an increase in the rate of anaerobic starch breakdown. Anaerobic NH₄⁺ assimilation also resulted in a two-fold increase in the rate of production of the major fermentative end-products in this alga, d-lactate and ethanol. There was no change in the rate of accumulation of the fermentative end product succinate but malate accumulated under anoxia during NH₄⁺ assimilation. A rapid increase in Gln and decline in Glu indicates that primary NH₄⁺ assimilation under anoxia was via glutamine synthetase-glutamate synthase. Almost all N assimilated under these conditions was sequestered in alanine. These results allow us to propose a model for the regulation of carbon metabolism during anaerobic NH₄⁺ assimilation.     

Regulation of rabbit liver fructose-1,6-bisphosphatase by metals, nucleotides, and fructose 2,6-bisphosphate as determined from fluorescence studies

The fluorescent nucleotide analogue formycin 5'-monophosphate (FMP) inhibits rabbit liver fructose-1,6-bisphosphatase (I50 = 17 microM, Hill coefficient = 1.2), as does the natural regulator AMP (I50 = 13 microM, Hill coefficient = 2.3), but exhibits little or no cooperativity of inhibition. Binding of FMP to fructose-1,6-bisphosphatase can be monitored by the increased fluorescence emission intensity (a 2.7-fold enhancement) or the increased fluorescence polarization of the probe. A single dissociation constant for FMP binding of 6.6 microM (4 sites per tetramer) was determined by monitoring fluorescence intensity. AMP displaces FMP from the enzyme as evidenced by a decrease in FMP fluorescence and polarization. The substrates, fructose 6-phosphate and fructose 1,6-bisphosphate, and inhibitors, methyl alpha-D-fructofuranoside 1,6-bisphosphate and fructose 2,6-bisphosphate, all increase the maximal fluorescence of enzyme-bound FMP but have little or no effect on FMP binding. Weak metal binding sites on rabbit liver fructose-1,6-bisphosphatase have been detected by the effect of Zn2+, Mn2+, and Mg2+ in displacing FMP from the enzyme. This is observed as a decrease in FMP fluorescence intensity and polarization in the presence of enzyme as a function of divalent cation concentration. The order of binding by divalent cations is Zn2+ = Mn2+ greater than Mg2+, and the Kd for Mn2+ displacement of FMP is 91 microM. Methyl alpha-D-fructofuranoside 1,6-bisphosphate, as well as fructose 6-phosphate and inorganic phosphate, enhances metal-mediated FMP displacement from rabbit liver fructose-1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light/Dark modulation of enzyme activity in developing barley leaves

Light/dark modulation of the ribulose-5-phosphate kinase, NADP⁺-glyceraldehyde-3-phosphate dehydrogenase, and fructose- 1,6-bisphosphatase activity was measured in the developing primary leaf of barley (Hordeum vulgare L.) seedlings. Ribulose-5-phosphate kinase and NADP⁺ -glyceraldehyde-3-phosphate dehydrogenase were fully light activated even at the earliest developmental stage sampled. In contrast, light modulation of fructose- 1,6-bisphosphatase exhibited a complex response to leaf developmental status. Light stimulation of fructose- 1,6-bisphosphatase activity (measured at pH 8.0) increased progressively during leaf development. On the other hand, acid fructose- 1,6-bisphosphatase activity (measured at pH 6.0) was inhibited by light, and this light inhibition was greater in the base of the leaf than in the tip of the leaf.     

“Sink” regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase in their cell wall involves a decrease of the Calvin-cycle enzymes and an increase of glycolytic enzymes

Leaves on transgenic tobacco plants expressing yeast-derived invertase in the apoplast develop clearly demarcated green and bleached sectors when they mature. The green areas contain low levels of soluble sugars and starch which are turned over on a daily basis, and have high rates of photosynthesis and low rates of respiration. The pale areas accumulate carbohydrate, photosynthesis is inhibited, and respiration increases. This provides a model system to investigate the sink regulation of photosynthetic metabolism by accumulating carbohydrate. The inhibition of photosynthesis is accompanied by a decrease of ribulose-1,5-bisphosphate and glycerate-3-phosphate, and an increase of triosephosphate and fructose-1,6-bisphosphate. The extracted activities of ribulose-1,5-bisphosphate carboxylase, fructose-1, 6-bisphosphatase and NADP-glyeraldehyde-3-phosphate dehydrogenase decreased. The activity of sucrose-phosphate synthase remained high or increased, an increased portion of the photosynthate was partitioned into soluble sugars rather than starch, and the pale areas showed few or no oscillations during transitions between darkness and saturating light in saturating CO₂. The increased rate of respiration was accompanied by an increased level of hexose-phosphates, triose-phosphates and fructose-1,6-bisphosphate while glycerate-3-phosphate and phosphoenolpyruvate decreased and pyruvate increased. The activities of pyruvate kinase, phosphofructokinase and pyrophosphate: fructose-6-phosphate phosphotransferase increased two- to four-fold. We conclude that an increased level of carbohydrate leads to a decreased level of Calvin-cycle enzymes and, thence, to an inhibition of photosynthesis. It also leads to an increased level of glycolytic enzymes and, thence, to a stimulation of respiration. These changes of enzymes are more important in middle- or long-term adjustments to high carbohydrate levels in the leaf than fine regulation due to depletion of inorganic phosphate or high levels of phosphorylated metabolites.Abbreviations Fru 1,6bisP fructose-1,6-bisphosphate - Fru 1,6bisPase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - Glc 1P glucose-1-phosphate - Glc6P glucose-6-phosphate - NADP-GAPDH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PFK phosphofructokinase - PEP phosphoenolpyruvate - PFP pyrophosphate:fructose-6-phosphate phosphotransferase - PGA glycerate-3-phosphate - PK pyruvate kinase - Pi inorganic phosphate - Ru1,5bisP ribulose-1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SPS sucrose-phosphate synthase - triose-P triose-phosphates     

Control of CO2 fixation regulation of stromal fructose-1,6-bisphosphatase in spinach by pH and Mg2+ concentration

The effect of pH and of Mg²⁺ concentration on the light activated form of stromal fructose-1,6-bisphosphatase (FBPase) was studied using the enzyme rapidly extracted from illuminated spinach chloroplasts. The (fructose-1,6-bisphosphate^4-)(Mg²⁺) complex has been identified as the substrate of the enzyme. Therefore, changes of pH and Mg²⁺ concentrations have an immediate effect on the activity of FBPase by shifting the pH and Mg²⁺ dependent equilibrium concentration of the substrate. In addition, changes of pH and Mg²⁺ concentration in the assay medium have a delayed effect on FBPase activity. A correlation of the activities observed using different pH and Mg²⁺ concentrations indicates, that the effect is not a consequence of the pH and Mg²⁺ concentration as such, but is caused by a shift in the equilibrium concentration of a hypothetical inhibitor fructose-1,6-bisphosphate^3- (uncomplexed), resulting in a change of the activation state of the enzyme. The interplay between a rapid effect on the concentration of the substrate and a delayed effect on the activation state enables a rigid control of stromal FBPase by stromal Mg²⁺ concentrations and pH. Fructose-1,6-bisphosphatase is allosterically inhibited by fructose-6-phosphate in a sigmoidal fashion, allowing a fine control of the enzyme by its product.Abbreviations Fru1,6 bis P fructose-1,6-bisphosphate - Fru6P fructose-6-phosphate - FBPase fructose-1,6-bisphosphatase Some of these results have been included in a preliminary report (Heldt et al. 1984)     

Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate.

Optimal conditions necessary for the reversible inactivation of crystalline rabbit muscle phosphofructokinase by homogeneous rabbit liver fructose-1,6-bisphosphatase have been studied. At higher enzyme levels (to 530 mug/ml of phosphofructokinase) the two proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HC1, 2 mM potassium phosphate, and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23, and 0 degree C. This inactivation did not require the presence of adenosine 5'-triphosphate or Mg2+ in the incubation mixture, but an adenosine 5'-triphosphate concentration of 2.7 mM or greater was required in the assay to keep phosphofructokinase in an inactive form. A mixture of activators (inorganic phosphate, (NH4)2SO4, and adenosine 5'-monophosphate), when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320 000 g of enzyme. Furthermore, the inactivation occurred in the absence of Mg2+ where the complete lack of fructose-1-6-bisphosphatase activity was confirmed directly. At lower phosphofructokinase concentrations (0.2-2 mug/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause inactivation. The inactivation did not occur when high concentrations of fructose 6-phosphate were present in the assay, or when the level of adenosine 5'-triphosphate was decreased. However, the inactivation was found at pH 8, where the effects of allosteric regulators on phosphofructokinase are greatly reduced. Experiments with rat liver phosphofructokinase showed that this enzyme was also subject to inhibition by rabbit liver fructose 1,6-bisphosphatase under conditions similar to those used in the muscle enzyme studies. Attempts to demonstrate direct interaction between phosphofructokinase and fructose-1,6-bisphosphate by physical methods were unsuccessful. Nevertheless, our results suggest that, under conditions which approximate the physiological state, the presence of fructose-1,6bisphosphatase can cause phosphofructokinase to assume an inactive conformation. This interaction may have a significant role in vivo in controlling the interrelationship between glycolysis and gluconeogenesis.     

Reduced-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana

(i) We have studied the influence of reduced phosphoglucose-isomerase (PGI) activity on photosynthetic carbon metabolism in mutants of Clarkia xantiana Gray (Onagraceae). The mutants had reduced plastid (75% or 50% of wildtype) or reduced cytosolic (64%, 36% or 18% of wildtype) PGI activity. (ii) Reduced plastid PGI had no significant effect on metabolism in low light. In high light, starch synthesis decreased by 50%. There was no corresponding increase of sucrose synthesis. Instead glycerate-3-phosphate, ribulose-1,5-bisphosphate, reduction of Q_A (the acceptor for photosystem II) and energy-dependent chlorophyll-fluorescence quenching increased, and O₂ evolution was inhibited by 25%. (iii) Decreased cytosolic PGI led to lower rates of sucrose synthesis, increased fructose-2,6-bisphosphate, glycerate-3-phosphate and ribulose-1,5-bisphosphate, and a stimulation of starch synthesis, but without a significant inhibition of O₂ evolution. Partitioning was most affected in low light, while the metabolite levels changed more at saturating irradiances. (iv) These results provide decisive evidence that fructose-2,6-bisphosphate can mediate a feedback inhibition of sucrose synthesis in response to accumulating hexose phosphates. They also provide evidence that the ensuing stimulation of starch synthesis is due to activation of ADP-glucose pyrophosphorylase by a rising glycerate-3-phosphate: inorganic phosphate ratio, and that this can occur without any loss of photosynthetic rate. However the effectiveness of these mechanisms varies, depending on the conditions. (v) These results are analysed using the approach of Kacser and Burns (1973, Trends Biochem. Sci. 7, 1149–1161) to provide estimates for the elasticities and flux-control coefficient of the cytosolic fructose-1,6-bisphosphatase, and to estimate the gain in the fructose-2,6-bisphosphate regulator cycle during feedback inhibition of sucrose synthesis.Abbreviations and symbols Chl chlorophyll - Fru6P fructose-6-phosphate - Frul,6bisP fructose-1,6-bisphosphate - Fru-1,6Pase fructose-1,6-bisphosphatase - Fru2,6bisP fructose-2,6-bisphosphate - Fru2,6Pase fructose-2,6-bisphosphatase - Glc6P glucose-6-phosphate - PGI phosphoglucose isomerase - Pi inorganic phosphate - Q_A acceptor for photosystem II - Ru1,5bisP ributose-1,5-bisphosphate - SPS sucrose-phosphate synthase     

Irreversible inactivation of Saccharomyces cerevisiae fructose-1,6-bisphosphatase independent of protein phosphorylation at Ser11

The fructose-1,6-bisphosphatase gene was used with multicopy plasmids to study rapid reversible and irreversible inactivation after addition of glucose to derepressed Saccharomyces cerevisiae cells. Both inactivation systems could inactivate the enzyme, even if 20-fold over-expressed. The putative serine residue, at which fructose-1,6-bisphosphatase is phosphorylated, was changed to an alanine residue without notably affecting the catalytic activity. No rapid reversible inactivation was observed with the mutated enzyme. Nonetheless, the modified enzyme was still irreversibly inactivated, clearly demonstrating that phosphorylation is an independent regulatory circuit that reduces fructose-1,6-bisphosphatase activity within seconds. Furthermore, irreversible glucose inactivation was not triggered by phosphorylation of the enzyme.