The effect of cyclic GMP on phosphofructokinase from rat tissues.

In view of the recently proposed hypothesis of biologic regulation through opposing influences of cyclic AMP and cyclic GMP, and since cyclic AMP is a well-known allosteric activator of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11), the effect of cyclic GMP on the activity of this enzyme from several rat tissues was investigated. It was found that cyclic GMP exerted an inhibitory effect on the activity of rat heart and skeletal muscle phosphofructokinase. This effect was most pronounced under conditions in which the enzyme was partially inhibited by ATP or by citrate. Cyclic GMP also antagonized the deinhibitory action of cyclic AMP and other allosteric activators, such as glucose 1,6-bisphosphate or AMP, on the ATP or citrate-inhibited heart or muscle phosphofructokinase. In contrast to the heart and skeletal muscle phosphofructokinase, the adipose-tissue enzyme was not affected by cyclic GMP to any significant degree. The antagonistic action of cyclic GMP to the activation of heart-phosphofructokinase, may suggest a mechanism by which the activity of phosphofructokinase is synchronized with the activity of glycogen phosphorylase, as a result of acetylcholine action in heart, to achieve a decrease in total glycogenolysis and glycolysis.     

The purine nucleotide cycle. Control of phosphofructokinase and glycolytic oscillations in muscle extracts.

Linked oscillations of the glycolytic pathway and the purine nucleotide cycle were studied in particle-free extracts of rat skeletal muscle. Under the conditions used, an accumulation of about 1 muM fructose diphosphate can trigger a sudden increase in phosphofructokinase activity. The activation by fructose diphosphate depends on the presence of AMP. When the AMP concentration drops, phosphofructokinase becomes inhibited, even though the fructose disphosphate concentration remains high. It is concluded that the oscillatory behavior can be of advantage for maintaining a high average [ATP]/[ADP] ratio.     

Control of phosphofructokinase from rat skeletal muscle. Effects of fructose diphosphate, AMP, ATP, and citrate.

Under conditions used previously for demonstrating glycolytic oscillations in muscle extracts (pH 6.65, 0.1 to 0.5 mM ATP), phosphofructokinase from rat skeletal muscle is strongly activated by micromolar concentrations of fructose diphosphate. The activation is dependent on the presence of AMP. Activation by fructose diphosphate and AMP, and inhibition by ATP, is primarily due to large changes in the apparent affinity of the enzyme for the substrate fructose 6-phosphate. These control properties can account for the generation of glycolytic oscillations. The enzyme was also studied under conditions approximating the metabolite contents of skeletal muscle in vivo (pH 7.0, 10mM ATP, 0.1 mM fructose 6-phosphate). Under these more inhibitory conditions, phosphofructokinase is strongly activated by low concentrations of fructose diphosphate, with half-maximal activation at about 10 muM. Citrate is a potent inhibitor at physiological concentrations, whereas AMP is a strong activator. Both AMP and citrate affect the maximum velocity and have little effect on affinity of the enzyme for fructose diphosphate.     

Properties of phsophofructokinase from the mucosa of rat jejunum and the relation to the lack of Pasteur effect

1. The properties of phosphofructokinase after its slight purification from the mucosa of rat jejunum were studied. 2. The enzyme is inhibited by almost 100% by an excess of ATP (1.6mm), with 0.2mm-fructose 6-phosphate. AMP, ADP, P(i) and NH(4) (+) at 0.2, 0.76, 1.0 and 2mm respectively do not individually prevent the inhibition of phosphofructokinase activity by 1.6mm-ATP with 0.2mm-fructose 6-phosphate to any great extent, but all of them together completely prevent the inhibition of phosphofructokinase by ATP. 3. One of the effects of high concentrations of ATP on the enzyme was to increase enormously the apparent K(m) value for the other substrate fructose 6-phosphate, and this increase is largely counteracted by the presence of AMP, ADP, P(i) and NH(4) (+). At low concentrations of ATP the above effectors individually decrease the concentration of fructose 6-phosphate required for half-maximum velocity and when present together they decrease it further, in a more than additive way. 4. When fructose 6-phosphate is present at a saturating concentration (5mm), 0.3mm-NH(4) (+) increases the maximum velocity of the reaction 3.3-fold; with 0.5mm-fructose 6-phosphate, 4.5mm-NH(4) (+) is required for maximum effect. The other effectors do not change the maximum reaction velocity. 5. The results presented here suggest that NH(4) (+), AMP, ADP and P(i) synergistically decrease the inhibition of phosphofructokinase activity at high concentrations of ATP by decreasing the concentration of fructose 6-phosphate required for half-maximum velocity. Such synergism among the effectors and an observed, low ;energy charge' [(ATP+(1/2)ADP)/(AMP+ADP+ATP)] in conjunction with the possibility of a relatively high NH(4) (+) and fructose 6-phosphate concentration in this tissue, may keep the mucosal phosphofructokinase active and uninhibited by ATP under aerobic conditions, thus explaining the high rate of aerobic glycolysis and the lack of Pasteur effect in this tissue.     

Regulation of glycolytic flux in an energetically controlled cell-free system: the effects of adenine nucleotide ratios, inorganic phosphate, pH, and citrate

A soluble extract from rat skeletal muscles has been used with purified mitochondrial ATPase (F₁) to develop steady states with respect to glycolytic flux, the concentrations of glycolytic intermediates and inorganic phosphate, and the concentrations and ratios of adenine nucleotides. Incubations were carried out in media resembling the ionic composition in the cell cytoplasm, in an attempt to evaluate the quantitative contributions of various effectors to the overall control mechanism under simulated in vivo conditions. The primary control reaction of glycolytic flux under the conditions studied could be identified with phosphofructokinase, followed by secondary control of the reaction catalyzed by hexokinase. Glycolytic flux was increased with increasing pH over the range 6.6–7.6, both in the absence and presence of ATPase. Without other added effectors, the glycolyzing extract maintained an ATP/ADP ratio of about 50 in the pH range 7.0–7.6, and phosphofructokinase was incompletely suppressed. Addition of increasing amounts of ATPase markedly stimulated glycolytic flux coincident with lowered steady-state ATP/ADP ratios, and decreased accumulation of hexose monophosphates. Control of flux by the ATP/ADP ratio (and simultaneously altered AMP concentration) was less effective if pH (7.3 to 7.6) or phosphate concentration (2 to 20 mm) was increased. Flux through phosphofructokinase was controlled principally when the ATP/ADP ratios were varied in the range between > 50 and 15. The inhibitory effect of citrate was evaluated. Suppression of glycolytic flux and accumulation of hexose monophosphates were dependent on incubation conditions. If the pH was 7.3 or less, and the phosphate concentration low (2 mm), flux through phosphofructokinase was significantly suppressed even at citrate concentrations less than 50 μm. Simultaneous decrease in the steady-state ATP/ADP ratio and elevation of AMP was ineffective in reversing this inhibition. At higher pH and, more dramatically, when the phosphate concentration was increased, sensitivity to citrate inhibition was markedly diminished. These data, taken together with studies of respiratory control with isolated mitochondria (21., 24.), J. Biol. Chem.250, 2275–2282) strongly suggest that adenine nucleotide control of both glycolysis and respiration is exerted when the ratio of free nucleotides (not protein bound) in the cytosol is in the range of 15 to > 50. The data further suggest that citrate plays an important role in the regulation of glycolysis in muscle when the ATP/ADP ratio is high (and the phosphate concentration is correspondingly low), but that this inhibition is overcome by liberation of inorganic phosphate during muscle contraction.     

The effects of ammonium, inorganic phosphate and potassium ions on the activity of phosphofructokinases from muscle and nervous tissues of vertebrates and invertebrates.

1. The effect of NH4+, Pi and K+ on phosphofructokinase from muscle and nervous tissues of a large number of animals was investigated. The activation of the enzyme from lobster abdominal muscle by NH4+ was increased synergistically by the presence of Pi or SO4(2-). In the absence of K+, NH4+ plus Pi markedly activated phosphofructokinase from all tissues studied. In the presence of 100 mM-K+, NH4+ plus Pi activated phosphofructokinase from nervous tissue and muscle of invertebrates and the enzyme from brain of vertebrates, but there was no effect of NH4+ plus Pi on the enzyme from the muscles of vertebrates. Nonetheless, NH4+ plus Pi increased the activity of vertebrate muscle phosphofructokinase in the presence of 50 mM-K+ at inhibitory concentrations of ATP, i.e. these ions de-inhibited the enzyme. In the absence of NH4+ plus Pi, K+ activated phosphofructokinase from vertebrate tissues at non-inhibitory ATP concentrations, but the effect was less marked with the enzyme from invertebrate tissues. Indeed, high concentrations of K+ (greater than 50 mM) caused inhibition of invertebrate tissue phosphofructokinase. Of the other alkali-metal ions tested, only Rb+ activated phosphofructokinase from lobster abdominal muscle and rat heart muscle. 2. The properties of lobster abdominal-muscle phosphofructokinase were studied in detail. This muscle was chosen as representative of invertebrate muscle because large quantities of tissue could be obtained from one animal and the enzyme was considerably more stable in tissue extracts than in extracts of insect flight muscle. In general, the properties of the enzyme from this tissue were similar to those of the enzyme from many other tissues: ATP concentrations above an optimum value inhibited the enzyme and this inhibition was decreased by raising the fructose 6-phosphate or the AMP concentration. In particular, NH4+ plus Pi activated the enzyme at noninhibitory concentrations of ATP and they also relieved ATP inhibition (see above). 3. It is suggested that increases in the concentration of NH4+ and Pi, under conditions of increased ATP utilization in certain muscles and/or nervous tissue, may play a part in the stimulation of glycolysis through the effects on phosphofructokinase (the effect may be a direct activation and/or a relief of ATP inhibition). Changes in the concentration of NH4+ and Pi are consistent with this theory in nervous tissue and the anaerobic type of muscles. The role of AMP deaminase in production of NH4+ from AMP in these tissues is discussed in relation to the control of glycolysis.     

Rat thyroid phosphofructokinase. Comparison of the regulatory and molecular properties with those of rat muscle enzyme.

The kinetic and molecular properties of rat thyroid phosphofructokinase (specific activity 134 units/mg) were compared with those of rat muscle phosphofructokinase (specific activity 135 units/mg). Thyroid and muscle phosphofructokinase showed similar sedimentation patterns in sucrose density gradients; their affinity for DEAE-cellulose was similar but not identical. A comparison of the kinetic properties revealed differences in the pH optima. Striking differences in the kinetic properties were shown below pH 7.4; the thyroid enzyme was less inhibited by ATP or citrate and more sensitive to activation by cyclic 3':5'-AMP than the muscle enzyme. A study of the effects of some cyclic as well as linear mononucleotides, such as cyclic AMP, cyclic IMP, cyclic GMP, cyclic CMP, cyclic UMP, 5'-AMP, and 3'-AMP on thyroid phosphofructokinase showed that at concentrations as low as 1 micrometer only cyclic AMP and cyclic IMP were able to activate thyroid enzyme in the presence of low fructose-6-P and high ATP concentrations.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase.The calculated cytosolic NAD⁺/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase and hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig.

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase. The calculated cytosolic NAD+/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Activation by phosphate of yeast phosphofructokinase.

The activity of yeast phosphofructokinase assayed in vitro at physiological concentrations of known substrates and effectors is 100-fold lower than the glycolytic flux observed in vivo. Phosphate synergistically with AMP activates the enzyme to a level within the range of the physiological needs. The activation by phosphate is pH-dependent: the activation is 100-fold at pH 6.4 while no effect is observed at pH 7.5. The activation by AMP, phosphate, or both together is primarily due to changes in the affinity of the enzyme for fructose-6-P. Under conditions similar to those prevailing in glycolysing yeast (pH 6.4, 1 mM ATP, 10 mM NH4+) the apparent affinity constant for fructose-6-P (S0.5) decreases from 3 to 1.4 mM upon addition of 1 mM AMP or 10 mM phosphate; if both activators are present together, S0.5 is further decreased to 0.2 mM. In all cases the cooperativity toward fructose-6-P remains unchanged. These results are consistent with a model for phosphofructokinase where two conformations, with different affinities for fructose-6-P and ATP, will present the same affinity for AMP and phosphate. AMP would diminish the affinity for ATP at the regulatory site and phosphate would increase the affinity for fructose-6-P. The results obtained indicate that the activity of phosphofructokinase in the shift glycolysis-gluconeogenesis is mainly regulated by changes in the concentration of fructose-6-P.     

Modifications in the allosteric properties of phosphofructokinase in rat erythrocytes and reticulocytes cross-linked with dimethyl suberimidate and 3,3'-dithiobispropionimidate

The kinetic behaviour of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been studied in situ, by using rat erythrocytes and reticulocytes treated with dimethyl suberimidate and 3,3'-dithiobispropionimidate as cross-linking reagents and with digitonin as the delipidating agent. Comparison of the ATP and fructose-6-P saturation curves of phosphofructokinase in dimethyl suberimidate-permeabilized cells with those obtained in haemolysates showed the enzyme to have reduced allosteric properties under in situ conditions, although it still responded to cyclic AMP (300 microM) added as allosteric effector. Non-sigmoidal fructose-6-P saturation curves were also observed using 3,3'-dithiobispropionimidate-permeabilized erythrocytes, either in the absence or in the presence of cyclic AMP. A hyperbolic behaviour was shown after cross-linking reversal of 3,3'-dithiobispropionimidate-permeabilized erythrocytes by treatment with dithiothreitol. Specific activity values of phosphofructokinase were always lower in permeabilized cells than in haemolysates. A significant inhibition of phosphofructokinase specific activity, without any effect on its allosteric behaviour, is exerted by reaction of dimethyl suberimidate or 3,3'-dithiobispropionimidate with erythrocyte lysates in the presence of an inhibitory concentration of ATP. These results suggest that penetration of the cross-linking reagent and its subsequent reaction with intracellular phosphofructokinase will have a direct effect upon the results obtained using this in situ approach.U     

The stimulus-secretion coupling of glucose-induced insulin release. Fasting-induced adaptation of key glycolytic enzymes in isolated islets.

The rate of glucose and fructose 6-phosphate phosphorylation in islet homogenates is reduced by prior fasting of the donor rats. In fed rats, the velocity of glucose phosphorylation at increasing glucose concentrations (0.1 to 100 mM) is compatible with the presence of two enzyme activities. A preferential effect of fasting upon the high Km enzyme activity can be documented either at low ATP concentration which enhances the fractional contribution of the high Km enzyme activity, or in the presence of glucose 6-phosphate, which suppresses the low Km enzyme activity. Islet phosphofructokinase activity was characterized by inhibition by citrate or high ATP concentrations, and relief from ATP inhibition by AMP. Fasting reduces the activity of phosphofructokinase without altering its sensitivity to ATP and AMP. Cyclic AMP fails to overcome the effect of fasting upon phosphofructokinase. The activity of phosphoglucoisomerase is unaffected by fasting. The fasting-induced adaptation of key glycolytic enzymes could account, in part at least, for reduced metabolism of glucose in islets from fasted rats.     

Complementarity in the regulation of phosphoglucomutase, phosphofructokinase and hexokinase; the role of glucose 1,6-bisphosphate.

ATP and citrate, the well known inhibitors of phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), were found to inhibit the activities of the multiple forms of phosphoglucomutase (alpha-D-glucose 1,6-bisphosphate: alpha-D-glucose 1-phosphate phosphotransferase, EC 2.7.5.1) from rat muscle and adipose tissue. This inhibition could be reversed by an increase in the glucose 1,6-bisphosphate (Glc-1,6-P2) concentration. Other known activators (deinhibitors) of phosphofructokinase, viz. cyclic AMP, AMP, ADP or Pi, had no direct deinhibitory action on the ATP or citrate inhibited multiple phosphoglucomutases. Cyclic AMP and AMP, could however lead indirectly to deinhibition of the phosphoglucomutases, by activating phosphofructokinase which catalyzes the ATP-dependent phosphorylation of glucose 1-phosphate to form Glc-1,6-P2, the la-ter then released the multiple phosphoglucomutases from ATP or citrate inhibition. The Glc-1,6-P2 was also found to exert a selective inhibitory effect on hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) type II, the predominant form in skeletal muscle. This selective inhibition by Glc-1,6-P2 was demonstrated on the multiple hexokinases which were resolved by cellogel electrophoresis or isolated by chromatography on DEAE-cellulose. Based on the in vitro studies it is suggested that during periods of highly active epinephrine-induced glycogenolysis in muscle, the Glc-1,6-P2, produced by the cyclic AMP-stimulated reaction of phosphofructokinase with glucose 1-phosphate, will release the phosphoglucomutases from ATP or citrate inhibition, and will depress the activity of muscle type II hexokinase.     

Etude de quelques propriétés de la phosphofructokinase érythrocytaire de l'homme normal

Human erythrocyte phosphofructokinase was purified 150 fold by DEAE cellulose adsorption and ammonium sulfate precipitation.At pH 7,5 the enzyme exhibits allosteric kinetics with respect to ATP, fructose 6 phosphate, and Mg²⁺.ATP at high concentration acted as an inhibitor and ADP, 5′AMP, 3′,5′, AMP, acted as activators. Both effectors seemed to decrease the homotropic interactions beetween the fructose 6 phosphate molecules.The activators increased the affinity of phosphofructokinase for the substrate (F6P), the inhibitor decreased it.These ligands had no effect on the maximum velocity of the reaction except in the case of ADP.Interactions between the substrates and the effector ligands on the enzyme were considered in terms of the Monod - Changeux - Wyman model for allosteric proteins.With GTP and ITP, no inhibition was observed. At saturing concentration of GTP, ATP still inhibited phosphofructokinase.Both 3′5′ AMP and fructose 6 phosphate increased the concentration of ATP required to produce an inhibition of 50 %.Citrate, like ATP, inhibited phosphofructokinase by binding most likely at the same allosteric site. Erythrocyte phosphofructokinase is inhibited by 2–3 DPG.The study of the relation log V max = f (pH) suggested, that the active center contains at least one imidazole and one sulfhydryl group.     

Phosphofructokinase in the liver fluke Fasciola hepatica. Purification and kinetic changes by phosphorylation

Phosphofructokinase from the liver fluke Fasciola hepatica was purified from extracts of the whole organisms. The molecular weight of the protomer as determined by sodium dodecyl sulfate-gel electrophoresis is 83,000. Phosphorylation of the liver fluke phosphofructokinase by the catalytic subunit of cAMP-dependent protein kinase occurred at a rate that was at least an order of magnitude greater than that observed with mammalian heart phosphofructokinase. The maximum level of phosphate incorporated was 0.22 mol P/mol of protomer. The kinetic properties of the enzyme were greatly altered as a result of phosphorylation. Compared to native enzyme, phosphorylated enzyme had a greater affinity for its substrate Fru-6-P and a decreased sensitivity to inhibition by ATP. These kinetic changes were similar to those of native enzyme in the presence of positive modifiers such as AMP. AMP also activated the phosphorylated enzyme. Activation of the phosphorylated enzyme by AMP was characterized by a further increase in affinity for Fru-6-P and a further decrease in sensitivity to ATP inhibition. Thus, the liver fluke phosphofructokinase can be modulated by covalent phosphorylation as well as noncovalent binding of different modifier ligands.     

The regulation of pea-seed phosphofructokinase by phosphoenolpyruvate

1. Pea-seed phosphofructokinase was purified 27-fold by a combination of fractionation with ethanol and ammonium sulphate. Under the conditions of assay, the enzyme was strongly inhibited by phosphoenolpyruvate. This inhibition was reversed by increasing the concentration of fructose 6-phosphate or magnesium chloride, or by lowering the ATP concentration. 2. Citrate, ADP and AMP inhibited phosphofructokinase and increased the sensitivity to phosphoenolpyruvate inhibition. Sulphate and inorganic phosphate stimulated the enzyme activity and decreased the sensitivity to phosphoenolpyruvate. 3. In the presence of inorganic phosphate and low concentrations of ATP, inhibition by phosphoenolpyruvate ceased and phosphoenolpyruvate became stimulatory. 4. The possible significance of these results in the control of plant carbohydrate metabolism is discussed.     

Purification and properties of adductor muscle phosphofructokinase from the oyster, Crassostrea virginica. The aerobic/anaerobic transition: role of arginine phosphate in enzyme control.

Phosphofructokinase from oyster (Crassostrea virginica) adductor muscle occurs in a single electrophorectic form at an activity of 8.1 mumol of product formed per minute per gram wet weight. The enzyme was purified to homogeneity by a novel method involving extraction in dilute ethanol and subsequent precipitation with polyethylene glycol. Oyster adductor phosphofructokinase has a molecular weight of 3400000 +/- 20000 as measured by Sephadex gel chromatography. Mg2+ or Mn2+ can satisfy the divalent ion requirement while ATP, GTP, or ITP can serve as phosphate donors for the reaction. Oyster adductor phosphofructokinase displays hyperbolic saturation kinetics with respect to all substrates (fructose 6-phosphate, ATP, and Mg2+) at either pH 7.9 OR PH 6.8. The Michaelis constant for fructose 6 phosphate at pH 6.8, the cellular pH of anoxic oyster tissues, is 3.5 mM. In the presence of AMP, by far the most potent activator and deinhibitor of the enzyme, this drops to 0.70 mM. Many traditional effectors of phosphofructokinase including citrate, NAD(P)H,Ca2+, fructose 1,6-bisphosphate, 3-phosphoglycerate, ADP, and phosphoenolpyruvate do not alter enzyme activity when tested at their physiological concentrations. Monovalent ions (K +, NH4+) are activators of the enzyme. ATP and arginine phosphate are the only compounds found to inhibit the adductor enzyme. The inhibitory action of both can be reversed by physiological concentrations of AMP(0.2- 1.0mM) and to a lesser extent by high concentrations of Pi (20 mM) and adenosine 3' :5'-monophosphate (0.1 mM). The two inhibitors exhibit very different pH versus inhibition profiles. The Ki (ATP) decreases from 5.0 mM to 1.3 mM as the pH decreases from 7.9 to 6.8, whereas the Ki for arginine phosphate increases from 1.3 mM to 4.5 mM for the same pH drop. Of all compounds tested, only AMP, within its physiological range, activated adductor phosphofructokinase significantly at low pH values. The kinetic data support the proposal that arginine phosphate, not ATP or citrate, is the most likely regulator of adductor phosphofructokinase in vivo under aerobic, high tissue pH, conditions. In anoxia, the depletion of arginine phosphate reserves and the increase in AMP concentrations in the tissue, coupled with the increase in the Ki for arginine phosphate brought about by low pH conditions, serves to activate phosphofructokinase to aid maintenance of anaerobic energy production.     

Activation of rabbit muscle phosphofructokinase by F-actin and reconstituted thin filaments

Striking effects of F-actin and the reconstituted thin filament of muscle on the catalytic activity of rabbit muscle phosphofructokinase are demonstrated through direct measurements of enzymatic activity by using the pH stat. The addition of F-actin to solutions of phosphofructokinase at low ionic strength (10 mM KCl and 5 mM MgCl2) partially reverses the inhibition of the enzyme seen at high ATP concentrations and increases the apparent affinity of the enzyme for fructose 6-phosphate with slight effect on Vmax. F-Actin augments the activation of the enzyme obtained with AMP and partially counters the inhibition obtained with citrate. The maximum effect in the reversal of ATP inhibition is about the same for combinations of either F-actin or the thin filament with AMP as it is for AMP alone. In general, the effect of F-actin on the catalytic activity of phosphofructokinase is larger than that of the thin filament. The activation of phosphofructokinase by F-actin persists at physiological ionic strength.     

Correlated changes of some enzyme activities and cofactor and substrate contents of pea cotyledon tissue during germination

1. The activities of six enzymes (hexokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, glucose 6-phosphate dehydrogenase and amylase) in extracts of pea cotyledons were determined. The activities during the first 10 days after germination showed individual and characteristic changes that indicate a specific control of both synthesis and destruction of enzymes. 2. Tissue contents of glucose, inorganic phosphate, glucose 6-phosphate, fructose 6-phosphate, ATP, ADP, AMP, NAD and NADP were also determined, and a correlation is reported between the substrate concentrations at day 1 and the subsequent enzymic activity. 3. The initial NAD(+)/NADH ratio value of 1 changed to about 3 by day 4; the NADP content was lower and changes in the oxidation state were less striking. The ratio of ATP to ADP and AMP remained virtually constant.     

A kinetic model for phosphofructokinase based on the paradoxical action of effectors.

Effectors of muscle phosphofructokinase show opposing action on the activity of the enzyme depending upon the concentration of phosphoryl donor employed in the assay. Established inhibitors, such as citrate, activate at low ATP or ITP concentrations while known activators, such as AMP, ADP, and cyclic AMP inhibit at low ATP or ITP concentrations. Inorganic phosphate, on the other hand, activates at all substrate concentrations. The paradoxical effects at low substrate concentrations are dependent upon the order of addition of reaction components. A model is proposed to explain these and other regulatory phenomena of phosphofructokinase.