Concentration Of Fructose 6-phosphate Research Articles

Dehydration during storage affects carbohydrate metabolism and the accumulation of non-sucrose carbohydrates in postharvest sugarbeet roots

Sugarbeet roots produced in cold climates are stored in large piles prior to processing and are cooled by ambient winter air. These storage conditions dehydrate roots, causing root respiration rate and sucrose loss in storage to increase. Dehydration likely alters carbohydrate metabolism due to a greater demand for respiratory substrates and may affect the accumulation of non-sucrose carbohydrates that impede processing. However, effects of dehydration on carbohydrate metabolism of postharvest sugarbeet roots have not been studied. Research, therefore, was conducted to determine the effect of mild and severe dehydration on concentrations of the non-sucrose carbohydrates, glucose, fructose, and raffinose, their biosynthetic precursors, and the concentrations of metabolites that are intermediates in respiratory sucrose catabolism through glycolysis and the TCA cycle. Roots were stored at 85 and 40% relative humidity for up to 28 d to generate mildly and severely dehydrated roots typical of roots in the interior and exterior of storage piles. Severe dehydration was associated with reductions in raffinose, galactinol, glucose 6-phosphate, fructose 6-phosphate, phosphoenolpyruvate, and pyruvate concentrations, and increases in inositol and malate concentrations, while mild dehydration was associated with increased raffinose concentration. Concentrations of other glycolytic and TCA cycle intermediates, as well as the invert sugars, glucose and fructose, were largely unaffected by dehydration. From these results, it is concluded that raffinose accumulation and biosynthesis is highly sensitive to root dehydration and that carbohydrate metabolism through glycolysis is altered by dehydration, presumably to provide increased substrates for respiration. Overall, this research provides new insight into the impact of environmental conditions within storage piles on raffinose accumulation and the mechanisms by which dehydration accelerates sucrose loss in stored sugarbeet roots. • Raffinose accumulates in roots stored under mildly dehydrating conditions. • Raffinose content declines in roots stored under severely dehydrating conditions. • Severe dehydration alters flux through glycolysis to support elevated respiration. • Invert sugar concentration is not significantly altered by dehydration in storage.

Use of 2-Photon Fluorescence Correlation Spectroscopy to Investigate Rat Liver Phosphofructokinase Self-Association

Phosphofructokinase (PFK) catalyzes the first committed step of glycolysis. The smallest active oligomer of PFK from rat liver (RLPFK) is a tetramer, however previous studies have demonstrated that at a physiological enzyme concentrations, fructose 6-phosphate (F6P) stabilizes species much larger than a tetramer. Using a Weber linkage argument, we propose that a highly associated species would demonstrate a higher affinity for F6P. 2-photon fluorescence correlation spectroscopy was used to quantify the oligomeric state of RLPFK in the presence of either F6P or MgATP. 11.6μM RLPFK labeled with fluorescein isothiocyanate, initially in the presence of F6P, was diluted to various concentrations from 116 nM to 1.5 nM into buffer with various concentrations, ranging from 3.2 mM to 0.2 mM, of either F6P and observed for 4 hours after dilution. In the presence of MgATP, 116nM RLPFK exists as a stable species with an apparent diffusion coefficient of 24μm2/sec, consistent with the value predicted for a tetramer. Diluting RLPFK to a concentration of 58nM or below in the presence of MgATP resulted in some dissociation into species smaller than the tetramer. The tetramer was stabilized by high [MgATP]. Dilution of RLPFK to 116nM in the presence of F6P gave an apparent diffusion coefficient of 8μM2/sec indicating a species much larger than a tetramer. This large species was stable over four hours in the presence of 3.2 to 0.8 mM F6P. At F6P concentrations of 0.4 mM and lower, RLPFK slowly dissociated into smaller species with an apparent diffusion coefficient of 16μM2/sec. The rate and extent of dissociation was dependent on both [RLPFK] and [F6P]. At low concentration of RLPFK and F6P the enzyme dissociates into species smaller than a tetramer. Funding: NIH-GM33216, NIH-CBI and Welch-A1543.

Glucuronoxylomannan from Cryptococcus neoformans Down-regulates the Enzyme 6-Phosphofructo-1-kinase of Macrophages

The encapsulated yeast Cryptococcus neoformans is the causative agent of cryptococosis, an opportunistic life-threatening infection. C. neoformans is coated by a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is considered a key virulence factor of this pathogen. The present work aimed at evaluating the effects of GXM on the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK). GXM inhibited PFK activity in cultured murine macrophages in both dose- and time-dependent manners, which occurred in parallel to cell viability decrease. The polysaccharide also inhibited purified PFK, promoting a decrease on the enzyme affinity for its substrates. In macrophages GXM and PFK partially co-localized, suggesting that internalized polysaccharide directly may interact with this enzyme. The mechanism of PFK inhibition involved dissociation of tetramers into weakly active dimers, as revealed by fluorescence spectroscopy. Allosteric modulators of the enzyme able to stabilize its tetrameric conformation attenuated the inhibition promoted by GXM. Altogether, our results suggest that the mechanism of GXM-induced cell death involves the inhibition of the glycolytic flux.

Mitochondrial Oxidative Phosphorylation Is Regulated by Fructose 1,6-Bisphosphate: A POSSIBLE ROLE IN CRABTREE EFFECT INDUCTION?

In numerous cell types, tumoral cells, proliferating cells, bacteria, and yeast, respiration is inhibited when high concentrations of glucose are added to the culture medium. This phenomenon has been named the "Crabtree effect." We used yeast to investigate (i) the short term event(s) associated with the Crabtree effect and (ii) a putative role of hexose phosphates in the inhibition of respiration. Indeed, yeast divide into "Crabtree-positive," where the Crabtree effect occurs, and "Crabtree-negative," where it does not. In mitochondria isolated from these two categories of yeast, we found that low, physiological concentrations of glucose 6-phosphate and fructose 6-phosphate slightly (20%) stimulated the respiratory flux and that this effect was strongly antagonized by fructose 1,6-bisphosphate (F16bP). On the other hand, F16bP by itself was able to inhibit mitochondrial respiration only in mitochondria isolated from a Crabtree-positive strain. Using permeabilized spheroplasts from Crabtree-positive yeast, we have shown that the sole effect observed at physiological concentrations of hexose phosphates is an inhibition of oxidative phosphorylation by F16bP. This F16bP-mediated inhibition was also observed in isolated rat liver mitochondria, extending this process to mammalian cells. From these results and taking into account that F16bP is able to accumulate in the cell cytoplasm, we propose that F16bP regulates oxidative phosphorylation and thus participates in the establishment of the Crabtree effect.

Identification of glucoselysine-6-phosphate deglycase, an enzyme involved in the metabolism of the fructation product glucoselysine

The metabolism of the glycation product fructose-epsilon-lysine in Escherichia coli involves its ATP-dependent phosphorylation by a specific kinase (FrlD), followed by the conversion of fructoselysine 6-phosphate into glucose 6-phosphate and lysine by fructoselysine-6-phosphate deglycase (FrlB), which is distantly related to the isomerase domain of glucosamine-6-phosphate synthase. As shown in the present work, several bacterial operons comprise: (1) a homologue of fructoselysine-6-phosphate deglycase; (2) a second homologue of the isomerase domain of glucosamine-6-phosphate synthase, more closely related to it; and (3) components of a novel phosphotransferase system, but no FrlD homologue. The FrlB homologue (GfrF) and the closer glucosamine-6-phosphate synthase homologue (GfrE) encoded by an Enterococcus faecium operon were expressed in E. coli and purified. Similar to FrlB, GfrF catalysed the reversible conversion of fructoselysine 6-phosphate into glucose 6-phosphate and lysine. When incubated with fructose 6-phosphate and elevated concentrations of lysine, GfrE catalysed the formation of a compound identified as 2-epsilon-lysino-2-deoxy-6-phospho-glucose (glucoselysine 6-phosphate) by NMR. GfrE also catalysed the reciprocal conversion, i.e. the formation of fructose 6-phosphate (but not glucose 6-phosphate) from glucoselysine 6-phosphate. The equilibrium constant of this reaction (0.8 M) suggests that the enzyme serves to degrade glucoselysine 6-phosphate. In conclusion, GfrF and GfrE serve to metabolize glycation products formed from lysine and glucose (fructoselysine) or fructose (glucoselysine), via their 6-phospho derivatives. The latter are presumably formed by the putative phosphotransferase system encoded by gfrA-gfrD. The designation gfr (glycation and fructation product degradation) is proposed for this operon. This is the first description of an enzyme participating in the metabolism of fructation products.

Aluminum-induced decrease in CO2 assimilation in citrus seedlings is unaccompanied by decreased activities of key enzymes involved in CO2 assimilation

'Cleopatra' tangerine (Citrus reshni Hort. ex Tanaka) seedlings were irrigated daily for 8 weeks with 1/4 strength Hoagland's nutrient solution containing 0 (control) or 2 mM aluminum (Al). Leaves from Al-treated plants had decreased CO2 assimilation and stomatal conductance, but increased intercellular CO2 concentrations compared with control leaves. On a leaf area basis, 2 mM Al increased activities of key enzymes in the Calvin cycle, including ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoribulokinase (PRK), stromal fructose-1,6-bisphosphatase (FBPase), and a key enzyme in starch synthesis, ADP-glucose pyrophosphorylase (AGPase), compared with control leaves. Aluminum had no effect on cytosolic FBPase activity, but it decreased sucrose phosphate synthase (SPS) activity. Aluminum had no effect on area-based concentrations of carbohydrates, glucose-6-phosphate (G6P) and fructose 6-phosphate (F6P) or the G6P:F6P ratio, but it decreased the area-based concentration of 3-phosphoglycerate (PGA). Photochemical quenching coefficient (qP) and electron transport rate through PSII were greatly reduced by Al. Non-photochemical quenching coefficient (NPQ) was less affected by Al than qP and electron transport rate through PSII. We conclude that the reduced rate of CO2 assimilation in Al-treated leaves was probably caused by a combination of factors such as reduced electron transport rate through PSII, increased closure of PSII reaction centers and increased photorespiration.

Antisense inhibition of sorbitol synthesis leads to up-regulation of starch synthesis without altering CO2 assimilation in apple leaves.

Sorbitol is a primary end-product of photosynthesis in apple (Malus domestica Borkh.) and many other tree fruit species of the Rosaceae family. Sorbitol synthesis shares a common hexose phosphate pool with sucrose synthesis in the cytosol. In this study, 'Greensleeves' apple was transformed with a cDNA encoding aldose 6-phosphate reductase (A6PR, EC 1.1.1.200) in the antisense orientation. Antisense expression of A6PR decreased A6PR activity in mature leaves to approximately 15-30% of the untransformed control. The antisense plants had lower concentrations of sorbitol but higher concentrations of sucrose and starch in mature leaves at both dusk and predawn. (14)CO(2) pulse-chase labeling at ambient CO(2) demonstrated that partitioning of the newly fixed carbon to starch was significantly increased, whereas that to sucrose remained unchanged in the antisense lines with decreased sorbitol synthesis. Total activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), sucrose-phosphate synthase (EC 2.4.1.14), and ADP-glucose pyrophosphorylase (EC 2.7.7.27) were not significantly altered in the antisense lines, whereas both stromal and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activities were higher in the antisense lines with 15% of the control A6PR activity. Concentrations of glucose 6-phosphate and fructose 6-phosphate (F6P) were higher in the antisense plants than in the control, but the 3-phosphoglycerate concentration was lower in the antisense plants with 15% of the control A6PR activity. Fructose 2, 6-bisphosphate concentration increased in the antisense plants, but not to the extent expected from the increase in F6P, comparing sucrose-synthesizing species. There was no significant difference in CO(2) assimilation in response to photon flux density or intercellular CO(2) concentration. We concluded that cytosolic FBPase activity in vivo was down-regulated and starch synthesis was up-regulated in response to decreased sorbitol synthesis. As a result, CO(2) assimilation in source leaves was sustained at both ambient CO(2) and saturating CO(2).

Estimation of binding constants for the substrate and activator of Rhodobacter sphaeroides adenosine 5 ′-diphosphate-glucose pyrophosphorylase using affinity capillary electrophoresis

Binding constants were determined for the activator fructose-6-phosphate (F6P) and substrate adenosine 5 ′-triphosphate (ATP) (in the presence and absence of F6P) to the recombinant wild-type (WT) Rhodobacter sphaeroides adenosine 5 ′-diphosphate-(ADP)-glucose pyrophosphorylase (ADPGlc PPase) using affinity capillary electrophoresis (ACE). In these binding studies, the capillary is initially injected with a plug of sample containing ADPGlc PPase and noninteracting standards. The sample is then subjected to increasing concentrations of F6P or ATP in the running buffer and electrophoresed. Analysis of the change in the migration times of ADPGlc PPase, relative to those of the noninteracting standards, as a function of the varying concentration of F6P or ATP yields a binding constant. The values obtained were in good agreement with kinetic parameters obtained from steady state activity assays. The method was extended to examine the F6P binding constants for the R33A and R22A enzymes and the ATP binding constants for the R8A enzyme in the presence and absence of F6P. The R33A enzyme has been shown by activity assays to be insensitive to F6P activation, indicating a defect in binding or in downstream transmission of the allosteric signal required for full activation. ACE indicated no apparent binding of F6P, supporting the former hypothesis. The R22A enzyme was shown by activity assays to have a ∼15-fold decrease in apparent affinity for F6P compared to that of WT while ACE indicated an affinity comparable to that of WT; potential reasons for this discrepancy are discussed. The R8A enzyme as measured by activity assays exhibits reduced fold-activation by F6P compared to that of WT but increased apparent affinity for ATP in the presence of F6P. The ACE results were in good agreement with the activity assay data, confirming the increased affinity for ATP in the presence of F6P. This method demonstrates the quantitative ability of ACE to study different binding sites/ligand interactions in allosteric enzymes.

Effects of a hexokinase II deletion on the dynamics of glycolysis in continuous cultures of

In glucose-limited aerobic chemostat cultures of a wild-type Saccharomyces cerevisiae and a derived hxk2 null strain, metabolic fluxes were identical. However, the concentrations of intracellular metabolites, especially fructose 1,6-bisphosphate, and hexose-phosphorylating activities differed. Interestingly, the hxk2 null strain showed a higher maximal growth rate and higher Crabtree threshold dilution rate, revealing a higher oxidative capacity for this strain. After a pulse of glucose, aerobic glucose-limited cultures of wild-type S. cerevisiae displayed an overshoot in the intracellular concentrations of glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate before a new steady state was established, in contrast to the hxk2 null strain which reached a new steady state without overshoot of these metabolites. At low dilution rates the overshoot of intracellular metabolites in the wild-type strain coincided with the immediate production of ethanol after the glucose pulse. In contrast, in the hxk2 null strain the production of ethanol started gradually. However, in spite of the initial differences in ethanol production and dynamic behaviour of the intracellular metabolites, the steady-state fluxes after transition from glucose limitation to glucose excess were not significantly different in the wild-type strain and the hxk2 null strain at any dilution rate.

Effects of a hexokinase II deletion on the dynamics of glycolysis in continuous cultures of Saccharomyces cerevisiae.

In glucose-limited aerobic chemostat cultures of a wild-type Saccharomyces cerevisiae and a derived hxk2 null strain, metabolic fluxes were identical. However, the concentrations of intracellular metabolites, especially fructose 1,6-bisphosphate, and hexose-phosphorylating activities differed. Interestingly, the hxk2 null strain showed a higher maximal growth rate and higher Crabtree threshold dilution rate, revealing a higher oxidative capacity for this strain. After a pulse of glucose, aerobic glucose-limited cultures of wild-type S. cerevisiae displayed an overshoot in the intracellular concentrations of glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate before a new steady state was established, in contrast to the hxk2 null strain which reached a new steady state without overshoot of these metabolites. At low dilution rates the overshoot of intracellular metabolites in the wild-type strain coincided with the immediate production of ethanol after the glucose pulse. In contrast, in the hxk2 null strain the production of ethanol started gradually. However, in spite of the initial differences in ethanol production and dynamic behaviour of the intracellular metabolites, the steady-state fluxes after transition from glucose limitation to glucose excess were not significantly different in the wild-type strain and the hxk2 null strain at any dilution rate.

Adaptation and maladaptation of the heart in diabetes: Part II: potential mechanisms.

The prevailing concept of the heart’s response to changes in its environment is a complex network of inter-connecting signal transduction cascades.1 In such a scheme, the focus is on communication of various cell surface receptors, heterotrimeric G-proteins, protein kinases, and transcription factors.2–4⇓⇓ Diabetes is a disorder of metabolic dysregulation. At first glance it appears that metabolism and the metabolic consequences of diabetes do not fit into this signal-response coupling scheme. Two questions arise. First, is metabolism simply an “effect” rather than a “cause” of adaptation? Second, is metabolism only a by-product of signal transduction-induced adaptation, allowing equilibrium (and therefore maintenance of function) in the presence of the other adaptational responses? An alternative is to take a new, less restricted view of metabolism. Beyond its stereotypical function as a provider of ATP, alterations in metabolic flux within the cell create essential signals for the adaptation of the heart to situations such as diabetes. This concept is novel for the heart, but has already been considered in the liver. Like the phosphorylation events occurring in signal transduction cascades, changes in metabolic flux are extremely rapid. For example, translocation of GLUT4 to the cell surface in response to insulin occurs within a second.5 We have previously found that increases or decreases in workload also change metabolic fluxes in seconds.6,7⇓ Therefore, changes in metabolites are rapid enough to allow them to act as signaling molecules. Many of these acute changes in metabolic flux are brought about by the same signal transduction cascades believed to be involved in the adaptation of the heart to changes in its environment. Phosphatidylinositol 3-kinase, Ca2+, and protein kinase C, all of which play a role in cardiac adaptation, regulate metabolism in the heart.8,9⇓ Metabolic signals therefore provide a …

ADP-dependent Glucokinase/Phosphofructokinase, a Novel Bifunctional Enzyme from the Hyperthermophilic Archaeon Methanococcus jannaschii

A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The gene encoded a protein of 462 amino acids with a molecular weight of 53,361. The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase. A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric. The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate. The K(m) values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mm, respectively. The K(m) values for ADP were 0.032 and 0.63 mm when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively. The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase. A gene duplication event might lead to the two enzymatic activities.

Apple leaf sucrose-phosphate synthase is inhibited by sorbitol-6-phosphate.

Sucrose-phosphate synthase (SPS) from mature apple (Malus domestica Borhk. cv. Gala) leaves was purified 34-fold to a final specific activity of 15.3 μmol mg-1 protein h-1. The enzyme showed hyperbolic saturation kinetics for both fructose-6-phosphate (F6P) (Km= 0.36 mM) and uridine-5'-diphosphoglucose (UDPG) (Km = 6.49 mM). Glucose-6-phosphate (G6P) was found to be an activator of apple SPS, and the activation was dependent upon the F6P concentration. At a concentration of 2 mM, G6P significantly decreased the Km for F6P and increased SPS activity. However, higher concentrations of G6P did not further stimulate SPS activity. In contrast to SPS from other plant species, inorganic phosphate (Pi) had little or no inhibitory effect on apple SPS. The apple leaf enzyme was inhibited 7-10% by 10 mM Pi when F6P concentrations were in the range of 2-10 mM. We observed that sorbitol-6-phosphate, an intermediate metabolite in sorbitol biosynthesis, was a competitive inhibitor of SPS with a Ki of 1.83 mM. Sorbitol-6-phosphate also inhibited G6P activation of SPS. Our results suggest that sucrose biosynthesis may be altered by the products of sorbitol biosynthesis in apple leaves.

The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus is a unique glycolytic enzyme that belongs to the cupin superfamily.

Pyrococcus furiosus uses a variant of the Embden-Meyerhof pathway during growth on sugars. All but one of the genes that encode the glycolytic enzymes of P. furiosus have previously been identified, either by homology searching of its genome or by reversed genetics. We here report the isolation of the missing link of the pyrococcal glycolysis, the phosphoglucose isomerase (PGI), which was purified to homogeneity from P. furiosus and biochemically characterized. The P. furiosus PGI, a dimer of identical 23.5-kDa subunits, catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate, with K(m) values of 1.99 and 0.63 mm, respectively. An optimum pH of 7.0 has been determined in both directions, and at its optimum temperature of 90 degrees C the enzyme has a half-life of 2.4 h. The N-terminal sequence was used for the identification of the pgiA gene in the P. furiosus genome. The pgiA transcription start site has been determined, and a monocistronic messenger was detected in P. furiosus during growth on maltose and pyruvate. The pgiA gene was functionally expressed in Escherichia coli BL21(DE3). The deduced amino acid sequence of this first archaeal PGI revealed that it is not related to its bacterial and eukaryal counterparts. In contrast, this archaeal PGI shares similarity with the cupin superfamily that consists of a variety of proteins that are generally involved in sugar metabolism in both prokaryotes and eukaryotes. As for the P. furiosus PGI, distinct phylogenetic origins have previously been reported for other enzymes from the pyrococcal glycolytic pathway. Apparently, convergent evolution by recruitment of several unique enzymes has resulted in the unique Pyrococcus glycolysis.

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 (Ka) 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 Ka of spinach leaf PFP for Fru-2,6-P2 was up to 2000-fold greater than that measured in the optimised assay and Vmax 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 Ka of spinach leaf PFP for Fru-1,6-P2 was approximately 50 microM. Physiological concentrations of PPi, 3PGA, PEP, ATP and Pi increased Ka up to 25-fold, and decreased Vmax by over 65%. From these results it was concluded that physiological concentrations of metabolites and Pi increase the Ka 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 Ka of PFP for Fru-1,6-P2 to an extent that activation of PFP by this compound is unlikely to be physiologically relevant.

Thermally induced changes in intracellular pH and modulators of phosphofructokinase in trout white muscle

The intracellular pH (pHi) and the concentrations of lactate and selected modulators of phosphofructokinase (PFK; EC 2.7.1.11) were measured in white epaxial muscle of 15 °C-acclimated rainbow trout (Oncorhynchus mykiss) maintained at 8, 15 or 22 °C for 48 h and sampled at rest and after 10 min of exhaustive exercise. The lactate accumulation resulting from exercise was 13 % smaller at 22 °C than at 8 and 15 °C. The estimated duration of burst performance was shorter at cold than at warm temperatures, whereas the average rate of lactate accumulation during burst performance was higher at 8 °C than at 15 and 22 °C. pHi rose when temperature decreased, but less than predicted by the imidazole alphastat hypothesis of Reeves. The effects of temperature on the pre-exercise concentrations of PFK modulators [adenylates, fructose 6-phosphate (F6P) and fructose 1,6-bisphosphate (FBP)] were generally negligible. In exhausted trout, adenylate concentrations were almost unaffected by temperature. In contrast, post-exercise FBP and F6P concentrations were significantly higher at low than at high temperatures. We interpret the response of F6P to temperature as an indication that the covariation of pHi and temperature is insufficient to prevent a cold-enhancement of PFK inhibition. Since F6P is a potent activator of PFK, we conclude that, in trout white muscle, thermally induced changes in F6P concentration probably help buffer the effects of temperature change on PFK activity.

Triosephosphate metabolism by mature boar spermatozoa.

Boar sperm rapidly interconverted dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, produced fructose-1,6-bisphosphate, approximately equilibrium concentrations of fructose 6-phosphate and glucose 6-phosphate but not glycerol or glycerol 3-phosphate. In the presence of 3-chloro-1-hydroxypropanone, an inhibitor of stage 2 of the glycolytic pathway, the triosephosphates were metabolized faster, produced less fructose-1,6-bisphosphate, fructose 6-phosphate and glucose 6-phosphate, but not glycerol or glycerol 3-phosphate. This suggests that these cells may have the capacity to convert glycolytic intermediates into a storage metabolite to conserve carbon atoms for the eventual synthesis of lactate.

Regulation of the supply of oxaloacetate for mitochondrial metabolism via phospho enol pyruvate carboxylase in barley leaf protoplasts II. Effects of metabolites on PEPC activity at different activation states of the protein

The regulation of the supply of oxaloacetate (OAA) for mitochondrial metabolism via phospho enol pyruvate carboxylase (PEPC) by metabolites is studied in barley ( Hordeum vulgare L.) leaf protoplasts in light or darkness as well as under photorespiratory or non-photorespiratory conditions. Measurements on PEPC activity were performed on samples quickly frozen in liquid nitrogen to break the cell and stop metabolism and thus preserve the in vivo activation state. Glycine, serine, pyruvate, acetyl-CoA, glycolate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and ADP had no significant effect on PEPC activity. Malate, aspartate and glutamate were strong inhibitors of PEPC activity decreasing the activity more in light versus darkness. However, at the physiological cytosolic concentration of these metabolites under the respective conditions, inhibition of PEPC activity was about the same with the exception of aspartate which inhibits more under non-photorespiratory than under photorespiratory conditions. 2-Oxoglutarate and glyoxylate decreased PEPC activity by 20 to 40% in the range of its physiological cytosolic concentration. Inhibition by physiological cytosolic concentrations of glutamine was limited. Glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglycerate, dihydroxyacetonphosphate and P i stimulated PEPC activity significantly in their physiological cytosolic concentration range. Physiological cytosolic concentrations of glucose 6-phosphate and fructose 6-phosphate activated PEPC activity to about the same extent under all conditions applied, while 3-phosphoglycerate and dihydroxyacetonphosphate stimulating stronger under non-photorespiratory versus photorespiratory conditions. Moreover, dihydroxyacetonphosphate stimulated PEPC activity more in light versus darkness under non-photorespiratory conditions. P i activation of PEPC activity decreases in light versus darkness under non-photorespiratory conditions. Stimulation of PEPC activity by citrate in its physiological concentration range is limited. Glucose 1-phosphate and AMP activated PEPC activity only at concentrations higher than their physiological levels in the cytosol. Determinations of PEPC activity in the presence of different malate/glucose 6-phosphate ratios revealed that glucose 6-phosphate totally relieved the inhibitory effect of malate. The regulatory properties of PEPC activity will be discussed in relation to its functions in C 3 plants.

Site-directed mutagenesis of rabbit muscle phosphofructokinase cDNA. Mutations at glutamine 200 affect the allosteric properties of the enzyme.

Full-length cDNA for rabbit muscle phosphofructokinase has been cloned and characterized (Li, J., Chen, Z., Lu, L., Byrnes, M., and Chang, S. H. (1990) Biochem. Biophys. Res. Commun. 170, 1056-1060). The 2.8-kilobase cDNA was inserted in the plasmid vector pPL2 and transformed into Escherichia coli cells deficient in endogenous phosphofructokinase activity (DF 1020). The recombinant phosphofructokinase so prepared is nearly identical in kinetic properties and size of subunits to the enzyme isolated from rabbit muscle. On the basis of the sequence homology between the muscle and the bacterial phosphofructokinases and the crystallographic structure of the latter, the glutamine at position 200 of the muscle enzyme is implicated in the allosteric transitions. This residue was replaced by alanine (Q200A), glutamate (Q200E), or arginine (Q200R). The purified enzymes were analyzed for quaternary structure, activity, and allosteric properties. The native and all the altered enzymes are tetramers. At pH 7.0, the wild-type enzyme is sensitive to inhibition by ATP at concentration above 0.6 mM, and its activity responds to fructose 6-phosphate concentration cooperatively at high ATP concentration. In contrast, the mutated enzyme Q200R is virtually insensitive to ATP inhibition up to 7 mM. Thus at high ATP concentration, its activity responds to fructose 6-phosphate concentration is a manner similar to the activated form of the native enzyme. Under the same conditions, mutant Q200E exhibits cooperative behavior only at much higher concentration of fructose 6-phosphate. Mutant Q200A is active at pH 8.0 but inactive at pH 7.0. The native enzyme and all three mutants are activated by inorganic phosphate and fructose 2,6-bisphosphate and inhibited by citrate.

Role of fructose 2,6-bisphosphate in the control of heart glycolysis

The aim of this work was to study whether changes in fructose 2,6-bisphosphate concentration are correlated with variations of the glycolytic flux in the isolated working rat heart. Glycolysis was stimulated to different extents by increasing the concentration of glucose, increasing the workload, or by the addition of insulin. The glycolytic flux was measured by the rate of detritiation of [2-3H]- and [3-3H]glucose. Under all the conditions tested, an increase in fructose 2,6-bisphosphate content was observed. The glucose- or insulin-induced increase in fructose 2,6-bisphosphate content was related to an increase in the concentration of fructose 6-phosphate, the substrate of 6-phosphofructo-2-kinase. An increase in the workload correlated with a 50% decrease in the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate. Similar changes in Km have been observed when purified heart 6-phosphofructo-2-kinase was phosphorylated in vitro by the cyclic AMP-dependent protein kinase or by the calcium/calmodulin-dependent protein kinase. Since the concentration of cyclic AMP was not affected by increasing the workload, it is possible that the change in Km of 6-phosphofructo-2-kinase, which was found in hearts submitted to a high load, resulted from phosphorylation by calcium/calmodulin protein kinase; other possibilities are not excluded. Anoxia decreased the external work developed by the heart, stimulated glycolysis and glycogenolysis, but did not increase fructose 2,6-bisphosphate.