Glycolytic activity in embryos of Pisum sativum and of non-dormant or dormant seeds of Avena sativa L. expressed through activities of PFK and PPi-PFK

A quantitative cytochemical assay for PPi-PFK activity in the presence of Fru-2,6-P2 is described along with its application to determine levels of activity in embryos of Pisum sativum and Avena sativa. The activity of ATP-PFK has also been studied in parallel as have PFK activities during the switch from dormant to non-dormant embryos in Avena sativa. PPi-PFK activity has been demonstrated in all tissues of Pisum sativum embryos and of Avena sativa embryos including the scutellum and the aleurone layers. The PPi-PFK activity was greater than that of ATP-PFK in both dormant and non-dormant seeds though with only marginally more activity in the dormant as opposed to the non-dormant state.     

Regulation and roles for alternative pathways of hexose metabolism in plants

Plant cells have two cytoplasmic pathways of glycolysis and gluconeogenesis for the reversible interconversion of fructose 6-phosphate (F-6-P) and fructose 1,6-bisphosphate (F-1,6-P₂). One pathway is described as a maintenance pathway that is catalyzed by a nucleotide triphosphate-dependent phosphofructokinase (EC 2.7.1.11; ATP-PFK) glycolytically and a F-1,6 bisphosphatase (EC 3.1.3.11) gluconeogenically. These are non-equilibrium reactions that are energy consuming. The second pathway, described as an adaptive pathway, is catalyzed by a readily reversible pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90; PP-PFK) in an equilibrium reaction that conserves energy through the utilization and the synthesis of pyrophosphate. A constitutive regulator cycle is also present for the synthesis and hydrolysis of fructose 2,6-bisphosphate (F-2,6-P₂) via a 2-kinase and a 2-phosphatase, respectively. The pathway catalyzed by the ATP-PFK and F-1,6-bisphosphatase, the maintenance pathway, is fairly constant in maximum activity in various plant tissues and shows less regulation by F-2,6-P₂. Plants use F-2,6-P₂ initially to regulate the adaptive pathway at the reversible PP_i-PFK step. The adaptive pathway, catalyzed by PP_i-PFK, varies in maximum activity with a variety of phenomena such as plant development or changing biological and physical environments. Plants can change F-2,6-P₂ levels rapidly, in less than 1 min when subjected to rapid environmental change, or change levels slowly over periods of hours and days as tissues develop. Both types of change enable plants to cope with the environmental and developmental changes that occur during their lifetimes. The two pathways of sugar metabolism can be efficiently linked by the cycling of uridylates and pyrophosphate required for sucrose breakdown via a proposed sucrose synthase pathway. The breakdown of sucrose via the sucrose synthase pathway requires half the net energy of breakdown via the invertase pathway. Pyrophosphate occurs in plant tissues as a substrate pool for biosynthetic reactions such as the PP_i-PFK or uridine diphosphate glucose pyrophosphorylase (EC 2.7.7.9; UDPG pyrophosphorylase) that function in the breakdown of imported sucrose. Also, pyrophosphate links the two glycolytic/gluco-neogenic pathways; and in a reciprocal manner pyrophosphate is produced as an energy source during gluconeogenic carbon flow from F-1,6-P₂ toward sucrose synthesis.     

Accumulation of fructose 1,6-bisphosphate in mutant cells of mucoid Pseudomonas aeruginosa as an evidence of phosphofructokinase activity

Phosphoglucose isomerase negative mutant of mucoid Pseudomonas aeruginosa accumulated relatively higher concentration of fructose 1,6-bisphosphate (Fru-1,6-P₂) when mannitol induced cells were incubated with this sugar alcohol. Also the toluene-treated cells of fructose 1,6-bisphosphate aldolase negative mutant of this organism produced Fru-1,6-P₂ from fructose 6-phosphate in presence of ATP, but not from 6-phosphogluconate. The results together suggested the presence of an ATP-dependent fructose 6-phosphate kinase (EC 2.7.1.11) in mucoid P. aeruginosa.Abbreviations ALD Fru-1,6-P₂ aldolse - DHAP dihydroxyacetone phosphate - F6P fructose 6-phosphate - G6P glucose 6-phosphate - Gly3P glyceraldehyde 3-phosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - PFK fructose 6-phosphate kinase - PGI phosphoglucose isomerase - 6PG 6-phosphogluconate     

PPi-Dependent Phosphofructokinase from Thermoproteus tenax,an Archaeal Descendant of an Ancient Line in Phosphofructokinase Evolution

Flux into the glycolytic pathway of most cells is controlled via allosteric regulation of the irreversible, committing step catalyzed by ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11), the key enzyme of glycolysis. In some organisms, the step is catalyzed by PP_i-dependent PFK (PP_i-PFK; EC 2.7.1.90), which uses PP_i instead of ATP as the phosphoryl donor, conserving ATP and rendering the reaction reversible under physiological conditions. We have determined the enzymic properties of PP_i-PFK from the anaerobic, hyperthermophilic archaeon Thermoproteus tenax, purified the enzyme to homogeneity, and sequenced the gene. The ∼100-kDa PP_i-PFK from T. tenax consists of 37-kDa subunits; is not regulated by classical effectors of ATP-PFKs such as ATP, ADP, fructose 2,6-bisphosphate, or metabolic intermediates; and shares 20 to 50% sequence identity with known PFK enzymes. Phylogenetic analyses of biochemically characterized PFKs grouped the enzymes into three monophyletic clusters: PFK group I represents only classical ATP-PFKs from Bacteria and Eucarya; PFK group II contains only PP_i-PFKs from the genus Propionibacterium, plants, and amitochondriate protists; whereas group III consists of PFKs with either cosubstrate specificity, i.e., the PP_i-dependent enzymes from T. tenax and Amycolatopsis methanolica and the ATP-PFK from Streptomyces coelicolor. Comparative analyses of the pattern of conserved active-site residues strongly suggest that the group III PFKs originally bound PP_i as a cosubstrate.As first discovered in Entamoeba histolytica (27), in some members of all three domains of life (Bacteria, Eucarya, and Archaea), the first committing step of glycolysis, the phosphorylation of fructose 6-phosphate (Fru 6-P), is catalyzed not by common ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11) but by an enzyme which uses PP_i as a phosphoryl donor (PP_i-PFK; EC 2.7.1.90) (22–34). The only archaeal PP_i-PFK described so far is the enzyme of Thermoproteus tenax, a hyperthermophilic, anaerobic archaeon which is able to grow chemolithotrophically with CO₂, H₂, and S⁰, as well as chemo-organothrophically in the presence of S⁰ and carbohydrates (11, 41). As shown by enzymatic and in vivo studies (pulse-labeling experiments), T. tenax metabolizes glucose mainly via a variation of the Embden-Meyerhof-Parnas pathway distinguished by the reversible PP_i-PFK reaction (34, 35).In contrast to the virtually irreversible reaction catalyzed by the ATP-PFK, the phosphorylation by PP_i is reversible. Thus, for thermodynamic reasons, the PP_i-PFK should be able to replace the enzymes of both the forward (ATP-PFK) and reverse (fructose-bisphosphatase [FBPase]) reactions. Consistent with the presumed bivalent function of the PP_i-dependent enzyme, in prokaryotes and parasitic protists which possess PP_i-PFK, little, if any, ATP-PFK or FBPase activity is present. Strikingly, the PP_i-PFKs of these organisms proved to be nonallosteric, suggesting that the control of the carbon flux through the pathway is no longer exerted by the PFK in these organisms. A considerably different situation has been described for higher plants and the green alga Euglena gracilis, showing comparable ATP-PFK, FBPase, and PP_i-PFK activities and allosteric regulation of their PP_i-dependent enzyme by fructose 2,6-bisphosphate (12, 22). However, in most cases it is not obvious which physiological role PP_i-PFK performs: reversible catalysis of glycolysis/gluconeogenesis, increase of the energy yield of glycolysis under certain conditions in which the energy charge is low, or ATP-conservation in obligately fermentative organisms (22).Closely related to questions concerning the biological function of PP_i-PFKs is the matter of their evolutionary origin: are these enzymes the result of a secondary adaptation from ATP-PFKs, or do they represent an original phenotype, as suggested by their specificity for PP_i, which is thought to be an ancient source of metabolic energy (9, 18, 19, 26). Indicated by sequence similarity (3, 4), all known PP_i- and ATP-PFKs are homologous and therefore originated from a common ancestral root. From more recent studies of Streptomyces coelicolor PFK (4), the previous assumption of a single event which separated PP_i- and ATP-PFKs had to be revised in favor of a multiple differentiation, leaving open, however, the question of the primary or secondary origin of PP_i-PFK.Understanding of PFK evolution has been impaired by a lack of knowledge concerning archaeal PFK, although the existence of ATP-PFK (31), PP_i-PFK (34), and also ADP-dependent PFK (16, 31) in Archaea has been described. To address the evolution of PFK, we describe the PP_i-PFK from T. tenax and compare its sequence and structure to those of known bacterial and eucaryal PFK enzymes.     

The expression of monocyte chemotactic protein (MCP-1) in human vascular endotheliumin vitro andin vivo

Total 6-phosphofructo-1-kinase (PFK) activity, amounts of each type of PFK subunit, and levels of fructose-2,6-P₂ in the cerebral cortex, midbrain, pons-medulla, and cerebellum of 3, 12, and 25 month rats were measured. Further, the role of fructose-2,6-P₂ in the regulation of brain PFK activity was examined. A positive correlation was found to exist between the reported losses of glucose utilization as measured by 2-deoxy-D-glucose uptake and PFK activity in each region. That is, both parameters decreased to their lowest level by 12 months of age and remained decreased and fairly constant thereafter. Fructose-2,6-P₂ levels did not appear to directly correlate with regional changes in glucose utilization. Also, region-specific and age-related alterations of the PFK subunits were found although these changes apparently did not correlate with decreased glucose utilization. Brain PFK is apparently saturated with fructose-2,6-P₂ due to the high endogenous levels, and it contains a large proportion of the C-type subunit which dampens catalytic efficiency. Consequently, brain PFK could exist in a conformational state such that it can readily consume fructose-6-P rather than in an inhibited state requiring activation. This may explain, in part, the ability of brain to efficiently but conservatively utilize available glucose in energy production.Abbreviations fructose-2,6-P₂ D-fructose 2,6-bisphosphate - fructose-6-P D-fructose 6-phosphate - PAGE Polyacrylamide Gel Electrophoresis - PFK 6-phosphofructo-1-kinase - PP_i-PFK Pyrophosphate-dependent Phosphofructokinase, ribose-1,5-P₂, ribose-1,5-bisphosphate - SDS Sodium Dodecyl Sulfate     

Citrate inhibition of rat-kidney cortex phosphofructokinase

The regulatory properties of citrate on the activity of phosphofructokinase (PFK) purified from rat-kidney cortex has been studied. Citrate produces increases in the K_0.5 for Fru-6-P and in the Hill coefficient as well as a decrease in the V_max of the reaction without affecting the kinetic parameters for ATP as substrate. ATP potentiates synergistically the effects of citrate as an inhibitor of the enzyme. Fru-2,6-P₂ and AMP at concentrations equal to K_a were not able to completely prevent citrate inhibition of the enzyme. Physiological concentrations of ATP and citrate produce a strong inhibition of renal PFK suggesting that may participate in the control of glycolysisin vivo.Abbreviations PFK 6-Phosphofructo-1-kinase (EC 2.7.1.11) - Fru-6-P Fructose 6-phosphate - Fru-2,6-P₂ Fructose 2,6-bisphosphate     

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)     

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

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

Thermotoga maritima phosphofructokinases: expression and characterization of two unique enzymes

A pyrophosphate-dependent phosphofructokinase (PP_i-PFK) and an ATP-dependent phosphofructokinase (ATP-PFK) from Thermotoga maritima have been cloned and characterized. The PP_i-PFK is unique in that the K_m and V_max values indicate that polyphosphate is the preferred substrate over pyrophosphate; the enzyme in reality is a polyphosphate-dependent PFK. The ATP-PFK was not significantly affected by common allosteric effectors (e.g., phosphoenolpyruvate) but was strongly inhibited by PP_i and polyphosphate. The results suggest that the control of the Embden-Meyerhof pathway in this organism is likely to be modulated by pyrophosphate and/or polyphosphate.     

Identification of two forms of PFK and a fructose-2,6-bisphosphate independent form of PFP in a green alga

Cell-free preparations from the green alga, Chlorella pyrenoidosa, contained two forms of phosphofructokinase (PFK), designated PFK I and PFK II. This represents the first evidence for a second form of PFK in green algae. A pyrophosphate D-fructose-6-phosphate, 1-phosphotransferase (PFP) activity, that was unaffected by the regulatory metabolite, fructose-2,6-bisphosphate, co-purified with PFK II through several steps. The data suggest that Chlorella pyrenoidosa resembles higher plants in containing two forms of PFK, but differs in containing an atypical form of PFP.Abbreviations PFK phosphofructokinase - PFP pyrophosphate D-fructose-6-phosphate, 1-phosphotransferase, Fru-2,6-P₂-fructose-2,6-bisphosphate - DEAE diethylaminoethyl-     

Role of fructose-1,6-bisphosphatase, fructose phosphotransferase, and phosphofructokinase in saccharide metabolism of four C3 grassland species under elevated CO2

We studied the effects of 15-months of elevated (700 μmol mol⁻¹) CO₂ concentration (EC) on the CO₂ assimilation rate, saccharide content, and the activity of key enzymes in the regulation of saccharide metabolism (glycolysis and gluconeogenesis) of four C₃ perennial temperate grassland species, the dicots Filipendula vulgaris and Salvia nemorosa and the monocots Festuca rupicola and Dactylis glomerata. The acclimation of photosynthesis to EC was downward in F. rupicola and D. glomerata whereas it was upward in F. vulgaris and S. nemorosa. At EC, F. rupicola and F. vulgaris leaves accumulated starch while soluble sugar contents were higher in F. vulgaris and D. glomerata. EC decreased pyrophosphate-D-fructose-6-phosphate l-phosphotransferase (PFP, EC 2.7.1.90) activity assayed with Fru-2,6-P₂ in F. vulgaris and D. glomerata and increased it in F. rupicola and S. nemorosa. Growth in EC decreased phosphofructokinase (PFK, EC 2.7.1.11) activity in all four species, the decrease being smallest in S. nemorosa and greatest in F. rupicola. With Fru-2,6-P2 in the assay medium, EC increased the PFP/PFK ratio, except in F. vulgaris. Cytosolic fructose-1,6-bisphosphatase (Fru-1,6-P₂ase, EC 3.1.3.11) was inhibited by EC, the effect being greatest in F. vulgaris and smallest in F. rupicola. Glucose-6-phosphate dehydrogenase (G6PDH EC 1.1.1.49) activity was decreased by growth EC in the four species. Activity ratios of Fru-1,6-P₂ase to PFP and PFK suggest that EC may shift sugar metabolism towards glycolysis in the dicots.     

The possible role of fructose 2,6-bisphosphate in the cyanide-mediated removal of embryonic dormancy in apple

The effects of pretreatment with HCN on the level of fructose 2.6-bisphosphate (F-2,6-P₂). on activity of fructose 6-phosphate 2-kinase (EC 2.7.1.150; F-6-P. 2K. enzyme synthesizing F-2,6-P₂), as well as on activities of PP₁-dependent and ATP-dependent phosphofructokinases (EC 2.7.1.90. PP₁-PFK and EC 2.7.1.11, ATP-PFK) were studied in cultured, dormant embryos of apple ( Malus domestica Borb. cv. Antonówka). HCN increased the F-2.6-P₂ level and F-6-P, 2K activity in embryonal axes (3-fold), but had no effect in cotyledons. HCN pretreatment of embryos or the addition of F-2.6-P₂ to enzyme extract stimulated PP₁, -PFK activity, whereas the activity of ATP-PFK was slightly inhibited by HCN in axes and in cotyledons. Glycolysis is one of the first processes in the germination of apple embryos, and the stimulation of glycolysis by HCN may be the result of F-6-P, 2K activation in the axes. This will lead to accumulation of F-2,6-P₂, which, in turn, enhances glycolysis by activation of PP₁PKF.     

On the mechanism of inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate

The inhibition of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) by fructose 2,6-bisphosphate (Fru-2,6-P₂) is shown to be competitive with the substrate, fructose 1,6-bisphosphate (Fru-1,6-P₂), with K_i for Fru-2,6-P₂ of approximately 0.5 μm. Binding of Fru-2,6-P₂ to the catalytic site is confirmed by the fact that it protects this site against modification by pyridoxal phosphate. Inhibition by Fru-2,6-P₂ is enhanced in the presence of a noninhibitory concentration (5 μm) of the allosteric inhibitor AMP and decreased by modification of the enzyme by limited proteolysis with subtilisin. Fru-2,6-P_2, unlike the substrate Fru-1,6-P₂, protects the enzyme against proteolysis by subtilisin or lysosomal proteinases.     

Physiological relevance of fructose 2,6-bisphosphate in the regulation of spinach leaf pyrophosphate:fructose 6-phosphate 1-phosphotransferase

A major problem in defining the physiological role of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) is the 1,000-fold discrepancy between the apparent affinity of PFP for its activator, fructose 2,6-bisphosphate (Fru-2,6-P₂), determined under optimum conditions in vitro and the estimated concentration of this signal metabolite in vivo. The aim of this study was to investigate the combined influence of metabolic intermediates and inorganic phosphate (Pi) on the activation of PFP by Fru-2,6-P₂. The enzyme was purified to near-homogeneity from leaves of spinach (Spinacia oleracea L.). Under optimal in vitro assay conditions, the activation constant (K _a) of spinach leaf PFP for Fru-2,6-P₂ in the glycolytic direction was 15.8 nM. However, in the presence of physiological concentrations of fructose 6-phosphate, inorganic pyrophosphate (PPi), 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), ATP and Pi the K _a of spinach leaf PFP for Fru-2,6-P₂ was up to 2000-fold greater than that measured in the optimised assay and V _max decreased by up to 62%. Similar effects were observed with PFP purified from potato (Solanum tuberosum L.) tubers. Cytosolic metabolites and Pi also influenced the response of PFP to activation by its substrate fructose 1,6-bisphosphate (Fru-1,6-P₂). When assayed under optimum conditions in the gluconeogenic direction, the K _a of spinach leaf PFP for Fru-1,6-P₂ was approximately 50 μM. Physiological concentrations of PPi, 3PGA, PEP, ATP and Pi increased K _a up to 25-fold, and decreased V _max by over 65%. From these results it was concluded that physiological concentrations of metabolites and Pi increase the K _a of PFP for Fru-2,6-P₂ to values approaching the concentration of the activator in vivo. Hence, measured changes in cytosolic Fru-2,6-P₂ levels could appreciably alter the activation state of PFP in vivo. Moreover, the same levels of metabolites increase the K _a of PFP for Fru-1,6-P₂ to an extent that activation of PFP by this compound is unlikely to be physiologically relevant. Received: 21 July 2000 / Accepted: 15 September 2000     

N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PP_i-dependent phosphofructokinase (TvPP_i-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPP_i-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to “short” bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.     

Reciprocal regulation of fructose 1,6-bisphosphatase and phosphofructokinase by fructose 2,6-bisphosphate in swine kidney

The effect of fructose 2,6-P₂, AMP and substrates on the coordinate inhibition of FBPase and activation of PFK in swine kidney has been examined. Fructose 2,6-P₂ inhibits the activity of FBPase and stimulates the activity of PFK in the presence of inhibitory concentrations of ATP. Under similar conditions 2.2 μM fructose 2,6-P₂ was required for 50% inhibition of FBPase and 0.04 μM fructose 2,6-P₂ restored 50% of the activity of PFK. Fructose 2,6-P₂ also enhanced the allosteric activation of PFK by AMP and it increased the extent of inhibition of FBPase by AMP. Fructose 2,6-P₂, AMP and fructose 6-P act cooperatively to stimulate the activity of PFK whereas the same latter two effectors and fructose 1,6-P₂ inhibit the activity of FBPase. Taken collectively, these results suggest that an increase in the intracellular level of fructose 2,6-P₂ during gluconeogenesis could effectively overcome the inhibition of PFK by ATP and simulataneously inactivate FBPase. When the level of fructose 2,6-P₂ is low, a glycolytic state would be restored, since under these conditions PFK would be inhibited by ATP and FBPase would be active.     

The subcellular location and characteristics of pyrophosphate-fructose-6-phosphate 1-phosphotransferase from suspension-cultured cells of soybean

The cytoplasm was identified as the probable location of pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) in suspension-cultured cells of soybean (Glycine max L.). The characteristics of the partially purified enzyme were investigated. The activity was strongly dependent on the presence of fructose 2,6-bisphosphate and this activator exerted its effects through a dramatic increase in the affinity of the enzyme for its substrates, fructose 6-phosphate and inorganic pyrophosphate. Saturation curves for all substrates were hyperbolic. The apparent molecular weight of the partially purified enzyme was 183000 by gel filtration chromatography and 128000 by sucrose-density-gradient centrifugation. The activation by fructose 2,6-bisphosphate was not accompanied by any measurable change in molecular weight. The possible role of this enzyme in the metabolism of non-photosynthetic sink tissues is discussed.Abbreviations PFP pyrophosphate-fructose-6-phosphate 1-phosphotransferase - P_i phosphate - PP_i pyrophosphate     

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

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

The role of phosphofructokinase in glycolytic control in the facultative anaerobe Sipunculus nudus

Summary The involvement of phosphofructokinase (PFK) in glycolytic control was investigated in the marine peanut worm Sipunculus nudus. Different glycolytic rates prevailed at rest and during functional and environmental anaerobiosis: in active animals glycogen depletion was enhanced by a factor of 120; during hypoxic exposure the glycolytic flux increased only slightly. Determination of the mass action ratio (MAR) revealed PFK as a non-equilibrium enzyme in all three physiological situations. Duirng muscular activity the PFK reaction was shifted towards equilibrium; this might account for the observed increase in glycolytic rate under these conditions. PFK was purified from the body wall muscle of S. nudus. The enzyme was inhibited by physiological ATP concentrations and an acidic pH; adenosine monophosphate (AMP), inorganic phosphate (P_i), and fructose-2,6-bisphosphate (F-2,6-P₂) served as activators. PFK activity, determined under simulated cellular conditions of rest and muscular work, agreed well with the glycolytic flux in the respective situations. However, under hypoxia PFK activity surpassed the glycolytic rate, indicating that PFK may not be rate-limiting under these conditions. The results suggest that glycolytic rate in S. nudus is mainly regulated by PFK during rest and activity. Under hypoxic conditions the regulatory function of PFK is less pronounced.Abbreviations ATP, ADP, AMP adenosine tri-, di-, monophosphate - DTT dithiothreitol - EDTA ethylene diaminetetra-acetic acid - F-6-P fructose-6-phosphate - F-1,6-P₂ fructose-1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate; bwm, body wall muscle; fresh mass, total body weight - G-6-P glucose-6-phosphate - H enthalpy change - K _a activation constant - K _eq equilibrium constant - K _i inhibition constant - K _m Michaelis constant - MAR mass action ratio - NMR nuclear magnetic resonance - PFK phosphofructokinase - P_i inorganic phosphate - PLA phospho-l-arginine - SD standard deviation - TRIS, TRIS (hydroxymethyl) aminomethane - TRA triethanolamine hydrochloride - V _max maximal velocity     

Mechanisms of glycolytic control during hibernation in the ground squirrel Spermophilus lateralis

Summary The mechanisms of glycolytic rate control during hibernation in the ground squirrel Spermophilus lateralis were investigated in four tissues: heart, liver, kidney, and leg muscle. Overall glycogen phosphorylase activity decreased significantly in liver and kidney to give 50% or 75% of the activity found in the corresponding euthermic organs, respectively. The concentration of fructose-2,6-bisphosphate (F-2,6-P₂) decreased significantly in heart and leg muscle during hibernation to 50% and 80% of euthermic tissue concentrations, respectively, but remained constant in liver and kidney. The overall activity of pyruvate dehydrogenase (PDH) in heart and kidney from hibernators was only 4% of the corresponding euthermic values. Measurements of phosphofructokinase (PFK) and pyruvate kinase (PK) kinetic parameters in euthermic and hibernating animals showed that heart and skeletal muscle had typical rabbit skeletal M-type PFK and M₁-type PK. Liver and kidney PFK were similar to the L-type enzyme from rabbit liver, whereas liver and kidney PK were similar to the M₂ isozyme found primarily in rabbit kidney. The kinetic parameters of PFK and PK from euthermic vs hibernating animals were not statistically different. These data indicate that tissue-specific phosphorylation of glycogen phosphorylase and PDH, as well as changes in the concentration of F-2,6-P₂ may be part of a general mechanism to coordinate glycolytic rate reduction in hibernating S. lateralis.Abbreviations ADP adenosine diphosphate - AMP adenosine monophosphate - ATP adenonine triphoshate - EDTA ethylenediaminetetra-acetic acid - EGTA ethylene glycol tetra-acetic acid - F-6-P fructose 6-phosphate - F-1,6-P₂ fructose 1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate - K _a activation coefficient - I₅₀ concentration of inhibitor which reduces control activity by 50% - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PFK 6-phosphofructo-1-kinase - PK pyruvate kinase