Phosphofructokinase from foot muscle of the whelk, Busycotypus canaliculatum: Evidence for covalent modification of the enzyme during anaerobiosis

Phosphofructokinase (PFK) was purified from foot muscle of aerobic and anaerobic (24 h of anoxia) whelks, Busycotypus canaliculatum. Fructose-6-P kinetics were sigmoidal at pH 7.0 with affinity constants, S_0.5, of 2.18 ± 0.10 (n_H = 2.5 ± 0.1) and 2.48 ± 0.13 mm (n_H = 2.7 ± 0.1) for the enzyme from aerobic versus anaerobic muscle. Affinity for ATP, like that for fructose-6-P, did not differ for the two enzymes (0.031 ± 0.003 for the aerobic vs 0.041 ± 0.007 mm for the anaerobic enzyme), but S_0.5 for Mg²⁺ was significantly different for the two enzymes (0.060 ± 0.006 vs 0.130 ± 0.020 mm). Whelk muscle PFK was activated by NH₄⁺, P_i, AMP, ADP, and fructose-2,6-P₂. NH₄⁺ and fructose-2,6-P₂ were less effective activators of PFK from anoxic muscle, with apparent K_a's 1.6- and 3.5-fold higher for the anaerobic vs aerobic enzyme. Activators decreased S_0.5 for fructose-6-P and reduced n_H. With the exception of fructose-2,6-P₂, the effects of activators on S_0.5 were the same for the enzyme from aerobic and anaerobic muscle; fructose-2,6-P₂ at 2.5 μm reduced S_0.5 by only 3.3-fold for the anaerobic enzyme compared to 5.5-fold for the aerobic enzyme. ATP was a strong substrate inhibitor of PFK; the enzyme from anaerobic muscle showed greater ATP inhibition, with I₅₀'s 1.5- to 2.0-fold lower than those for the aerobic enzyme. The kinetic differences between PFK from anaerobic versus aerobic foot muscle (stronger ATP inhibition and decreased sensitivity to activators for the anaerobic enzyme) were consistent with kinetic differences reported for the phosphorylated versus dephosphorylated forms, respectively, of PFK in other systems. Treatment of PFK from anaerobic muscle with alkaline phosphatase resulted in a decrease in the K_a for fructose-2,6-P₂ to a level similar to that of the aerobic enzyme. The physiological stress of anoxia may, therefore, induce a covalent modification of PFK.     

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.     

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     

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     

Fructose-1-phosphate-6-sulfate as an alternative substrate for aldolase and fructose-1,6-diphosphatase.

Fructose-1-phosphate-6-sulfate was prepared by direct sulfurylation of fructose, and selective phosphorylation of the 6-sulfuryl isomer by phosphofructokinase. The ketose derivative was used as a substrate for aldolase and fructose-1,6-diphosphatase. Kinetic studies with aldolase showed that the alternative substrate binds one third as well as fructose-1,6-P₂ yet 900 fold greater than fructose-1-P. The V_m was intermediate between the two ketose phosphates. From kinetic studies with skeletal muscle fructose-1,6-diphosphatase at pH 7.5 a K_m of 8 μM and a V_m approximately 6% that for fructose-1,6-P₂ was obtained.     

High resolution phosphorus NMR spectroscopy of transfer ribonucleic acids

Summary A new activator of phosphofructokinase, which is bound to the enzyme and released during its purification, has been discovered. Its structure has been determined as -D Fructose-2,6-P₂ by chemical synthesis, analysis of various degradation products and NMR. D-Fructose-2,6-P₂ is the most potent activator of phosphofructokinase and relieves inhibition of the enzyme by ATP and citrate. It lowers the K_m for fructose-6-P from 6 mM to 0.1 mM.Fructose-6-P,2-kinase catalyzes the synthesis of fructose-2,6-P₂ from fructose-6-P and ATP, and the enzyme has been partially purified. The degradation of fructose-2,6-P₂ is catalyzed by fructose-2,6-bisphosphatase. Thus a metabolic cycle could occur between fructose-6-P and fructose-2,6-P₂, which are catalyzed by these two opposing enzymes. The activities of these enzymes can be controlled by phosphorylation. Fructose-6-P,2-kinase is inactivated by phosphorylation catalyzed by either cAMP dependent protein kinase or phosphorylase kinase. The inactive, phospho-fructose-6-P,2-kinase is activated by dephosphorylation catalyzed by phosphorylase phosphatase. On the other hand, fructose-2,6-bisphosphatase is activated by phosphorylation catalyzed by cAMP dependent protein kinase.Investigation into the hormonal regulation of phosphofructokinase reveals that glucagon stimulates phosphorylation of phosphofructokinase which results in decreased affinity for fructose-2,6-P₂, and decreases the fructose-2,6-P₂ levels. This decreased level in fructose-2,6-P₂ appears to be due to the decreased synthesis by inactivation of fructose-2,6-P₂,2-kinase and increased degradation as a result of activation of fructose-2,6-bisphosphatase. Such a reciprocal change in these two enzymes has been demonstrated in the hepatocytes treated by glucagon and epinephrine. The implications of these observations in respect to possible coordinated controls of glycolysis and glycogen metabolism are discussed.     

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     

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     

Properties of Pyrophosphate:Fructose-6-Phosphate Phosphotransferase from Endosperm of Developing Wheat (Triticum aestivum L.) Grains

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90) from endosperm of developing wheat (Triticum aestivum L.) grains was purified to apparent homogeneity with about 52% recovery using ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose and gel filtration through Sepharose-CL-6B. The purified enzyme, having a molecular weight of about 170,000, was a dimer with subunit molecular weights of 90,000 and 80,000, respectively. The enzyme exhibited maximum activity at pH 7.5 and was highly specific for pyrophosphate (PPi). None of the nucleoside mono-, di- or triphosphate could replace PPi as a source of energy and inorganic phosphate (Pi). Similarly, the enzyme was highly specific for fructose-6-phosphate. It had a requirement for Mg²⁺ and exhibited hyperbolic kinetics with all substrates including Mg²⁺. K_m values as determined by Lineweaver-Burk plots were 322, 31, 139, and 129 micromolar, respectively, for fructose-6-phosphate, PPi, fructose-1,6-bisphosphate and Pi. Kinetic constants were determined in the presence of fructose-2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for its substrates. Initial velocity studies indicated kinetic mechanism to be sequential. At saturating concentrations of fructose-2,6-bisphosphate (1 micromolar), Pi strongly inhibited PFP; the inhibition being mixed with respect to both fructose-6-phosphate and PPi, with K_i values of 0.78 and 1.2 millimolar, respectively. The inhibition pattern further confirmed the mechanism to be sequential with random binding of the substrates. Probable role of PFP in endosperm of developing wheat grains (sink tissues) is discussed.     

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     

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-     

Sucrose metabolism in developing endosperm of immature wheat (Triticum aestivum L.) grain

With a view to investigating the role of the enzyme pyrophosphate-fructose-6-phosphate-1-phosphotransferase (PFP) in sucrose breakdown in developing endosperm of wheat grain, the activity of PFP and related enzymes such as phosphofructokinase (PFK), fructose-6-bisphosphatase (FBPase), fructose-6-phosphate-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (F2, 6-P2ase) and the contents of the various intermediates of the pathway serving either the substrate or the effectors of these enzymes such as glu-6-P,glu-1-P,fru-6-P,fru-1,6-P2,DHAP,G3P, UDP-glucose, ADP-glucose, Pi,PPi and fru-2,6-P2 have been determined at 5 days intervals starting from day-5 after anthesis until day-40 after anthesis. These enzymes except PFK-2 had their peak activity at day-25 after anthesis. The activity of PFP was several fold higher than that of PFK at each stage of grain development. PFK-2 exhibited the lowest activity. The various intermediates again had their maximum concentration either at day-20 or day-25 after anthesis. Among hexose phosphates studied, glu-6-P was present in highest concentration at each stage of grain development. The level of Pi was much higher than those of PPi and fru-2,6-P2. Similarly, concentration of UDP-glucose was higher than that of ADP-glucose. Based on these results, it is proposed that the major role of the enzyme PFP in developing wheat grain is to provide PPi for sucrose breakdown via sucrose synthase.     

Reassessment of an apparent hyperactive form of phosphofructokinase from plants

In the assay of phosphofructokinase (PFK) from endosperm of germinating castor bean (Ricinus communis L.) there is a transient stimulation of initial activity by fructose 2,6-bisphosphate. This activation is due to metabolism of a limited amount of pyrophosphate (a contaminant of commercial ATP) by PPi:fructose 6-phosphate phosphotransferase (PFP), which is present in the extract. Both this activity and the amount of pyrophosphate contamination are sufficient to account for the initial increase in apparent PFK activity. The transient burst of activity is dependent on both of the above factors. Based on studies of a similar hyperactive PFK, others have proposed that PFK and PFP may be interconverted (Balogh et al. 1984 FEBS Lett 169: 287-292). The evidence for such conversions is reinterpreted in the context of the current results.     

Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana ( Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (alpha) and 60-kDa (beta) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi ( K(m) values = 32, 9.7, 25, and 410 microM, respectively) in the presence of saturating (5 microM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity ( K(a)=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5-6.0 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP ( K(m)=34 and 21 microM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P ( S0.5=540 and 90 microM, respectively). Allosteric effects of phospho enolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II ( I50=2.3 and 900 microM, respectively), while Pi activated PFK I by reducing its sensitivity to PEP inhibition. Our results are consistent with: (i) the respiratory climacteric being regulated by fine (allosteric) control of pre-existing enzymes; and (ii) primary and secondary glycolytic flux control being exerted at the levels of PEP and Fru 6-P metabolism, respectively.     

Pyrophosphate Fructose-6-P 1-Phosphotransferase from Tomato Fruit : Evidence for Change during Ripening

Three forms of pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP) were purified from both green and red tomato (Lycopersicon esculentum) fruit: (a) a classical form (designated Q₂) containing α- (66 kilodalton) and β- (60 kilodalton) subunits; (b) a form (Q₁) containing a β-doublet subunit; and (c) a form (Q₀) that appeared to contain a β-singlet subunit. Several lines of evidence suggested that the different forms occur under physiological conditions. Q₂ was purified to apparent electrophoretic homogeneity; Q₁ and Q₀ were highly purified, but not to homogeneity. The distribution of the PFP forms from red (versus green) tomato was: Q₂, 29% (90%); Q₁, 47% (6%); and Q₀, 24% (4%). The major difference distinguishing the red from the green tomato enzymes was the fructose-2,6-bisphosphate (Fru-2,6-P₂)-induced change in K_m for fructose-6-phosphate (Fru-6-P), the `green forms' showing markedly enhanced affinity on activation (K<sub>m</sub> decrease of 7-9-fold) and the `red forms' showing either little change (Q₀, Q₁) or a relatively small (2.5-fold) affinity increase (Q₂). The results extend our earlier findings with carrot root to another tissue and indicate that forms of PFP showing low or no affinity increase for Fru 6-P on activation by Fru-2,6-P₂ (here Q₁ and Q₀) are associated with sugar storage, whereas the classical form (Q₂), which shows a pronounced affinity increase, is more important for starch storage.     

Cytosolic phosphofructokinase from spinach leaves : I. Purification, characteristics, and regulation

Cytosolic ATP-dependent phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was enriched 2600-fold by (NH₄)₂SO₄ fractionation, DEAE anion exchange chromatography, Blue Sepharose CL-6B, and ATP agarose type 3-affinity chromatography. The final preparation had a specific activity of 417 nkat per milligram protein and exhibited four bands between 50 and 70 kilodaltons following denaturing electrophoresis. Only one band of ATP- and fructose 6-phosphate (F-6-P)-dependent, Pistimulated activity was detected following isoelectric focusing PAGE and nondenaturing discontinuous PAGE of the final preparation. Crude extracts contained, in addition to the band observed in the final preparation, a second band that was inhibited by Pi. The latter band is presumably chloroplastic PFK. PFK was stimulated by the anions Pi²⁻, Cl⁻, SO₄²⁻, NO₃⁻, HAsO₄²⁻, and HCO₃⁻ but was not affected by NH₄⁺. Pi and Mg²⁺ changed the response of PFK toward pH and affected the saturation kinetics of F-6-P. In general, activity was highest when Pi was high and (or) Mg²⁺ was low. Phosphoenolpyruvate (PEP), 2-PGA, and PPi, but not 3-PGA, inhibited PFK. Although the inhibition by PEP and 2-PGA was reduced or relieved by Pi, the inhibition by PPi was not affected by Pi. F-2, 6-P₂ had no effect upon the activity of PFK. It is proposed that, in the cytosol of spinach leaves, PFK is likely to be more active during the dark, when cytosolic Pi levels are high, than in the light.     

The catalytic direction of pyrophosphate:fructose 6-phosphate 1-phosphotransferase in rice coleoptiles in anoxia

The catalytic direction of pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP; EC 2.7.1.90) in coleoptiles of rice ( Oryza sativa L.) seedlings subjected to anoxia stress is discussed. The stress greatly induced ethanol synthesis and increased activities of alcohol dehydrogenase (ADH; EC 1.1.1.1) and pyruvate decarboxylase (PDC; EC 4.1.1.1) in the coleoptiles, whereas the elevated PDC activity was much lower than the elevated ADH activity, suggesting that PDC may be one of the limiting factors for ethanolic fermentation in rice coleoptiles. Anoxic stress decreased concentrations of fructose 6-phosphate (Fru-6-P) and glucose 6-phosphate, and increased concentration of fructose 1,6-bisphosphate (Fru-1,6-bisP) in the coleoptiles. PFP activity in rice coleoptiles was low in an aerobic condition and increased during the stress, whereas no significant increase was found in ATP:fructose-6-phosphate 1-phosphotransferase (PFK; EC 2.7.1.11) activity in stressed coleoptiles. Fructose 2,6-bisphosphate concentration in rice coleoptiles was increased by the stress and pyrophosphate concentration was above the K_m for the forward direction of PFP and was sufficient to inhibit the reverse direction of PFP. Under stress conditions the potential of carbon flux from Fru-6-P toward ethanol through PFK may be much lower than the potential of carbon flux from pyruvate toward ethanol through PDC. These results suggest that PFP may play an important role in maintaining active glycolysis and ethanolic fermentation in rice coleoptiles in anoxia.     

Physiological relevance of the changing subunit composition and regulatory properties of the 6-phosphofructo-1-kinase isozyme pools during heart and muscle development

During postnatal development, the subunit compositions of the 6-phosphofructo-l-kinase isozyme pools of heart and skeletal muscle are known to change. The isozyme pools from fetal muscle were composed of the L-type (60%), and M-type (36%) and C-type (4%) subunits and the isozymes from fetal and early neonatal heart contain nearly equal amounts of all three subunits. During postnatal development of both tissues, the proportion of the M-type subunit increases until it is the only type present in adult muscle and the major subunit in adult heart (7507o). The isozyme pool from fetal muscle exhibit a decreased affinity for fructose-6-P and a greater susceptibility to ATP inhibition compared to the M-rich isozymes which are subsequently present. The isozyme pools from fetal and early neonatal heart, if compared to the M-rich isozymes which are present later during heart development and to the fetal muscle isozymes, exhibited the least affinity for fructose-6-P and the greatest susceptibility to ATP inhibition. Comparison of the isozyme pools containing little or no C-type subunit with those from fetal and early neonatal heart clearly indicates that the presence of substantial levels of the C-type subunit imposed a decreased ability for fructose-2,6-P₂ to both lower affinity for fructose-6-P and antagonize sensitivity to ATP inhibition. Although still not thoroughly appreciated, it appears that the changing nature of the isozyme pools in these tissues permits regulation of glucose metabolism in a manner which allows efficient utilization of nutritional opportunities and which adequately meets the energy requirements of each tissue at different stages of development.Abbreviations PFK 6-phosphofructo-l-kinase - fructose-6-P D-fructose-6-phosphate - fr-t_ose-2,6-P₂ D-fructose-2,6-bisphosphate     

Purification and characterization of phosphofructokinase of Bacillus licheniformis

The phosphofructokinase (PFK) of Bacillus licheniformis was purified about 50–65-fold and examined for a number of enzymatic and physical characteristics. The enzyme is quite unstable under normal assay conditions, but Mg²⁺, K⁺, adenosine-5′-diphosphate, phosphoenolpyruvate (PEP), and fructose-6-phosphate (fru-6-P) are fairly effective stabilizing agents. Saturation functions for ATP and fru-6-P were hyperbolic. Several attempts to induce positive cooperative binding of fru-6-P were unsuccessful. However, “sigmoidal” saturation kinetics for fru-6-P could be observed under assay conditions that permitted an irreversible inactivation of the PFK during assay. Several divalent cations could support the catalysis of B. licheniformis PFK and the enzyme was activated by both NH₄⁺ and K⁺ ions. B. licheniformis PFK is inhibited by citrate, ATP, PEP, Ca²⁺, and several other metabolic intermediates, but the inhibition caused by citrate and ATP at high fru-6-P concentration and by calcium can be relieved by Mg²⁺ addition while PEP inhibition is specifically relieved by fru-6-P. There are at least three binding sites for PEP on the PFK molecule. The active form of this PFK has a molecular weight of about 134,000 daltons. In the presence of Mg²⁺, adenosine-5′-triphosphate (ATP), and PEP, at 0 °C, the PFK molecule is rapidly dissociated to an inactive form with a molecular weight of about 68,000 daltons. Association of these subunits to yield the active form of PFK occurs spontaneously, and rapidly, when the temperature is raised to 30 °C. Ninety percent of the original activity is recovered after activation. Growth of B. licheniformis on several different substrates resulted in minor variations of PFK activity. In a parallel fashion, sporulation involved no irreversible inactivation of PFK and the level of the activity was about the same throughout the life cycle. Control of this enzyme during sporulation could be affected by any or all of the cell constituents found to regulate PFK activity in vitro, but it is considered likely that the most significant in vivo negative effector is PEP, with this inhibition being reversed by fru-6-P.     

Binding and regulatory properties of phosphofructokinase from swine kidney

Summary The influence of fructose 2,6-bisphosphate on the activation of purified swine kidney phosphofructokinase as a function of the concentration of fructose 6P, ATP and citrate was investigated. The purified enzyme was nearly completely inhibited in the presence of 2 mM ATP. The addition of 20 nM fructose 2,6-P₂ reversed the inhibition and restored more than 80% of the activity. In the absence of fructose 2,6-P₂ the reaction showed a sigmoidal dependence on fructose 6-phosphate. The addition of 10 nM fructose 2,6-bisphosphate decreased the K_0.5 for fructose 6-phosphate from 3 mM to 0.4 mM in the presence of 1.5 mM ATP. These results clearly show that fructose 2,6-bisphosphate increases the affinity of the enzyme for fructose 6-phosphate and decreases the inhibitory effect of ATP. The extent of inhibition by citrate was also significantly decreased in the presence of fructose 2,6-phosphate.The influence of various effectors of phosphofructokinase on the binding of ATP and fructose 6-P to the enzyme was examined in gel filtration studies. It was found that kidney phosphofructokinase binds 5.6 moles of fructose 6-P per mole of enzyme, which corresponds to about one site per subunit of tetrameric enzyme. The K_D for fructose 6-P was 13 µM and in the presence of 0.5 mM ATP it increased to 27 µM. The addition of 0.3 mM citrate also increased the K_D for fructose 6-P to about 40 µM. AMP, 10 µM, decreased the K_D to 5 µM and the addition of fructose 2,6-phosphate decreased the K_D for fructose 6-P to 0.9 µM. The addition of these compounds did not effect the maximal amount of fructose 6-P bound to the enzyme, which indicated that the binding site for these compounds might be near, but was not identical to the fructose 6-P binding site. The enzyme bound a maximum of about 12.5 moles of ATP per mole, which corresponds to 3 moles per subunit. The K_D of the site with the highest affinity for ATP was 4 µM, and it increased to 15 µM in the presence of fructose 2,6-bisphosphate. The addition of 50 µM fructose 1,6-bisphosphate increased the K_D for ATP to 5.9 µM. AMP increased the K_D to 5.9 µM whereas 0.3 mM citrate decreased the K_D for ATP to about 2 µM. The K_D for AMP, was 2.0 µM; the K_D for cyclic AMP was 1.0 µM; the K_D for ADP was 0.9 µM; the K_D for fructose 1,6-bisphosphate was 0.5 µM; the K_D for citrate was 0.4 µM and the K_D for fructose 2,6-bisphosphate was about 0.1 µM. A maximum of about 4 moles of AMP, ADP and cyclic AMP and fructose 2,6-bisphosphate were bound per mole of enzyme. Taken collectively, these and previous studies (9) indicate that fructose 2,6-phosphate is a very effective activator of swine kidney phosphofructokinase. This effector binds to the enzyme with a very high affinity, and significantly decreases the binding of ATP at the inhibitory site on the enzyme.