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PubMed1993

Interaction of phosphofructokinase with tubulin and microtubules. Quantitative evaluation of the mutual effects.

The linked equilibria involved in the binding of phosphofructokinase (EC 2.7.1.11, ATP:D-fructose-6-phosphate 1-phosphotransferase) to tubulin and microtubules were studied at high ionic strength in vitro. The concentration-dependent dissociation of phosphofructokinase was analyzed in the absence and presence of tubulin or microtubules, and the binding of kinase to the tubulin dimer and microtubules was compared. Enzyme activity of phosphofructokinase was inhibited by both tubulin and microtubules: the relative inhibition increased with decreasing enzyme concentration. The complex formation between phosphofructokinase and tubulin was demonstrated by means of fluorescent anisotropy. Concentration-dependent copelleting of the kinase with taxol-stabilized microtubules revealed binding of the enzyme to microtubules as well as phosphofructokinase-enhanced pelleting of microtubules. The binding data agree with the enzyme kinetic findings that the inactive dissociated forms of phosphofructokinase (monomer-dimer) are involved in the heterologous complex formation. Microtubule reorganization (bundle formation) by phosphofructokinase was established by turbidity measurements and sedimentation experiments. The binding data are consistent with a simple molecular model for the interactions in phosphofructokinase-tubulin/microtubules systems.

Animals↗
PubMed1999

Purification and properties of the pyrophosphate-dependent phosphofructokinase from Dictyoglomus thermophilum Rt46 B.1.

The distribution of phosphofructokinase phosphoryl donor subtypes (ATP-, ADP-, and pyrophosphate) in the deeply rooted phylogenetic lineages of thermophiles is of interest with regard to the evolution of phosphofructokinase activity and of the Embden-Meyerhof pathway. In this article we present the first biochemical description of a thermostable pyrophosphate-dependent phosphofructokinase from the hyperthermophilic bacterium Dictyoglomus thermophilum. The enzyme was not allosterically controlled by traditional modulators of phosphofructokinases and has significant activity with tripolyphosphate and polyphosphate. Kinetic parameters of the enzyme suggest it plays primarily a glycolytic role. The enzyme required Mg2+ for optimal activity, was partially activated by some monovalent and divalent cations, and was strongly inhibited by Cu2+. The sequence of the 21 N-terminal residues suggests that the enzyme is most similar to the pyrophosphate-dependent phosphofructokinases from Amycolatopsis methanolica and the hyperthermophilic crenarchaeon Thermoproteus tenax, enzymes which have been suggested to represent an ancient lineage of phosphofructokinases (Siebers et al. 1998). The unexpected finding of a pyrophosphate-dependent phosphofructokinase in Dictyoglomus thermophilum, which is phylogenetically related to Thermotoga maritima, previously shown to possess an ATP-dependent phosphofructokinase activity, is discussed.

Amino Acid Sequence↗
PubMed1983

Role of AMP deaminase reaction in the response of phosphofructokinase to the adenylate energy charge.

The role of NH+4 ion and AMP deaminase reaction in the activation of phosphofructokinase with respect to its response to the adenylate energy charge was investigated using permeabilized yeast cells. (a) Phosphofructokinase and AMP deaminase were activated by the decrease in the adenylate energy charge. The addition of NH+4 further stimulated the phosphofructokinase activity in the presence of intracellular level of K+, and the optimal energy charge value giving the maximal response of the enzyme was shifted from 0.3 to the value above 0.5. (b) The increase in NH+4 ion produced through the activation of AMP deaminase by spermine which shows no direct action on the phosphofructokinase activity can activate phosphofructokinase with shift of the optimal energy charge value of the enzyme to 0.5 in the presence of K+, whereas the optimal energy charge value for AMP deaminase reaction was not affected by the addition of spermine. Phosphofructokinase can be activated most effectively by the physiological decrease in the energy charge under the condition of increased NH+4 in the presence of K+. The possibility that the interaction of phosphofructokinase with AMP deaminase under hypoxic condition might be a contributing factor to the Pasteur effect is discussed.

AMP Deaminase↗
PubMed1984

Metabolite-induced activation of hepatic phosphofructokinase.

Hepatic phosphofructokinase, isolated in a medium containing 100 mM (NH4)2SO4, can be activated by ATP. This metabolite-induced activation was investigated in view of the suggestion that it is related to phosphorylation of phosphofructokinase. The results obtained do not support this interpretation. Inhibitors of protein phosphatases (NaF) and kinases (the Mg++-chelator, ethylene diamine tetraacetic acid) did not affect the recovery of phosphofructokinase. In contrast, media of high ionic strength reduced the phosphofructokinase activity and rendered the enzyme sensitive to ATP-induced activation. Activation was also induced by other known effectors of phosphofructokinase (nucleoside triphosphates, fructose bisphosphates) and was not dependent on Mg++-ions. It is suggested that activation represents ligand-induced reversal of the inactivation of phosphofructokinase which occurs at high ionic strength. The differential sensitivity of phosphofructokinase from fed or starved animals to inactivation and reactivation is discussed.

Adenosine Triphosphate↗
PubMed1983

A liver-type mutation in a case of pronounced erythrocyte phosphofructokinase deficiency without clinical expression.

A marked erythrocyte phosphofructokinase deficiency was detected in a healthy man. His enzymatic activity was only 25% that of normal controls. His father and his son had erythrocytic phosphofructokinase activities of 50-55% that of normal controls. The chromatographic separation of erythrocytic phosphofructokinase isozymes, as well as immunological studies revealed a decrease in L-type phosphofructokinase activity. The lowered erythrocytic L-type phosphofructokinase activity was not accompanied by a decreased level of L-type phosphofructokinase in proteins. The L/M subunit ratio was similar to that of normal subjects. The defect resulted from the synthesis of stable L-type mutant subunit with high electrophoretic mobility. White blood cells, which synthesize mostly the same isozyme as L-type phosphofructokinase also showed a decreased activity and a high electrophoretic mobility. In spite of this important deficiency, and of significant metabolic alterations (a slight decrease in ATP; 2,3-diphosphoglycerate; triose phosphate), hemolysis did not appear in the propositus.

Antigen-Antibody Complex↗
PubMed1996

Myofibril-bound muscle phosphofructokinase is less sensitive to inhibition by ATP than the free enzyme, but retains its sensitivity to stimulation by bisphosphorylated hexoses.

Phosphofructokinase activity is modulated by allosteric effectors and macromolecular interactions (e.g. binding to myofibrillar components). The aim of this study was to determine the effects of ATP and bisphosphorylated sugars upon phosphofructokinase in the presence of myofibrils. Myofibrils were prepared from resting and electrically stimulated rat muscle. Dephosphorylation of myofibrils was performed with alkaline phosphatase acid. Purified rabbit skeletal muscle phosphofructokinase was used for all experiments. Myofibrils from resting muscle showed a higher capacity to bind phosphofructokinase and a lower phosphate content than myofibrils from stimulated muscle. Dephosphorylation of myofibrils did not increase their binding capacity. Myofibrils greatly counteracted the inhibition of phosphofructokinase by high concentrations of ATP, without affecting maximum activity. In the presence of myofibrils, both glucose 1,6-bisphosphate and fructose 2,6-bisphosphate additionally activated muscle phosphofructokinase. We suggest that the binding of phosphofructokinase to myofibrils in combination with increasing glucose 1,6-bisphosphate concentration could be important in the enhancement of the glycolytic flux that takes place during muscle contraction.

Adenosine Triphosphate↗
PubMed2000

Quantitative characterization of homo- and heteroassociations of muscle phosphofructokinase with aldolase.

Dissociation of purified phosphofructokinase accompanied with inactivation was analyzed in the absence and presence of aldolase and the data were compared with those obtained with muscle extract. The kinetics of the decrease in enzymatic activity was highly dependent on the dilution factor in both cases, but the inactivation appeared to be biphasic only with extract. The inactivation of the phosphofructokinase was impeded by addition of excess of aldolase. Time courses of kinase inactivation were fitted by alternative kinetic models to characterize the multiple equilibria of several homo- and hetero-oligomers of phosphofructokinase. The combination of modeling data obtained with purified and extract systems suggests that aldolase binds to an intermediate dimer of phosphofructokinase and within this heterocomplex the kinase is completely active. The intermediate dimer is stabilized by association with microtubules and the kinase activity decreased due to dilution can be recovered by addition of excess aldolase. In extract, the phosphofructokinase is of sigmoidal character (Hill coefficient of 2.3); the addition of excess exogenous aldolase to phosphofructokinase resulted in heterocomplex formation displaying Michaelian kinetics. The possible physiological relevance of heterocomplex formation of phosphofructokinase in muscle extract is discussed.

Allosteric Regulation↗
PubMed1991

Protein-induced inactivation and phosphorylation of rabbit muscle phosphofructokinase.

Several previously untested proteins promote the reversible inactivation of rabbit skeletal muscle phosphofructokinase. Grouped in decreasing order of effectiveness, they include the following: skeletal muscle troponin C greater than troponin, the two smooth muscle myosin light chains, alpha-actinin, and S-100 much greater than parvalbumin and soybean trypsin inhibitor. The efficiency of troponin C in this process may even exceed that previously reported for calmodulin. Sequences near calcium binding site III are apparently involved in the troponin C-phosphofructokinase interaction. Troponin C and calmodulin exert calcium-dependent effects on the physical and chemical properties of muscle phosphofructokinase. When calcium is present, comigration with either protein allows the enzyme to enter the stacking gel during urea-polyacrylamide gel electrophoresis. Both enhance the phosphorylation of phosphofructokinase catalyzed by the cAMP-dependent protein kinase, with phosphate incorporations approaching 2 mol of P/mol of protomer. Reaction occurs at Ser774 and at Ser376--a novel site whose phosphorylation is highly sensitive to troponin C and less so to calmodulin. Maximum phosphorylation has slight effect on the catalytic activity of the enzyme under standard assay conditions. The troponin C induced or calmodulin-induced phosphorylation of phosphofructokinase requires calcium and is strongly inhibited by either fructose 2,6-bisphosphate or fructose 1,6-bisphosphate. Inactivation occurs in the presence or absence of calcium, with generally higher concentrations of effectors required for protection in the latter case. Liver and yeast phosphofructokinases shows little activity loss in the presence of either calmodulin or troponin C. We have developed and tested a general mathematical model for the protein-induced inactivation of phosphofructokinase which may find application to other systems.

Amino Acid Sequence↗
PubMed1997

Characterization of microtubule-phosphofructokinase complex: specific effects of MgATP and vinblastine.

Phosphofructokinase interacts with both microtubules and microtubules containing microtubule-associated proteins to produce bundling and periodical cross-bridging of tubules. Immunoelectron microscopy using anti-phosphofructokinase antibodies provided direct evidence that the kinase molecules are responsible for the cross-bridging of microtubules. Limited proteolysis by subtilisin, a procedure that cleaves the N-terminal segment of the free enzyme as well as the C-terminal "tails" of tubulin subunits exposed on microtubules, showed that while phosphofructokinase becomes resistant, tubulin retains sensitivity against proteolysis within the heterologous complex. These data suggest that the N-terminal segment of the enzyme, but not the C-terminal "tail" of tubulin subunits, is involved in the interaction between the microtubule and the kinase. The phosphorylation of phosphofructokinase or microtubules containing microtubule-associated proteins by the cAMP-dependent protein kinase did not interfere with the heterologous complex formation. MgATP prevents phosphofructokinase binding to the microtubules, and it can displace the enzyme from the single microtubules. However, the bundled microtubules are apparently resistant to the MgATP dissociation effect. Modelling of the assembly process suggests that the tubulin-kinase complex is able to polymerize as the free tubulin. Vinblastine, an anti-mitotic agent, inhibits tubulin assembly; however, its inhibitory effect is partially suppressed in the presence of phosphofructokinase. Fluorescence anisotropy measurements indicated that kinase and vinblastine compete for tubulin binding with no evidence for ternary complex formation. This competitive mechanism and the ability of the tubulin-enzyme complex to polymerize into microtubules may result in the resistance of the tubulin-enzyme complex against the inhibition of assembly induced by vinblastine. Microtubules formed in the presence of vinblastine plus phosphofructokinase can be visualized by electron microscopy. A molecular model is suggested that summarizes the effects of MgATP and vinblastine on the multiple equilibria in the tubulin/microtubules/phosphofructokinase system.

Adenosine Triphosphate↗
PubMed1984

The effect of glucose on the activity of phosphofructokinase in the mucosa of rat small intestine.

In common with other phosphofructokinase isoenzymes, phosphofructokinase in the epithelial cells of rat small-intestinal mucosa is activated by fructose 2,6-bisphosphate. However, fructose 2,6-bisphosphate was found not to be present in mucosa as judged by three criteria: (1) chromatography on Sephadex G-25 of crude mucosal extracts from fed rats did not result in a decrease, or indeed any change, in the activity of phosphofructokinase under suboptimal conditions at pH7; (2) ultrafiltrates of mucosal extracts did not possess any acid-labile activating activity when tested against chromatographed liver phosphofructokinase; (3) phosphofructokinase-2 activity was not detectable in mucosal extracts. Furthermore, the perfusion in vitro of isolated loops of jejunum or the incubation of mucosal scrapings from either fed rats or rats starved for 48 h showed that the activity of mucosal phosphofructokinase is not subject to short-term regulation by glucose. These observations are consistent with the view that phosphofructokinase is the rate-limiting enzyme of glycolysis in intestinal mucosa and account for the fact that the rate of glucose utilization by rat small intestine is not very responsive to changes in the concentration of glucose in the lumen.

Animals↗
PubMed1976

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.

Adenosine Monophosphate↗
PubMed2004

Effects of insulin and actin on phosphofructokinase activity and cellular distribution in skeletal muscle.

In this work, we report evidences that the association of phosphofructokinase and F-actin can be affected by insulin stimulation in rabbit skeletal muscle homogenates and that this association can be a mechanism of phosphofructokinase regulation. Through co-sedimentation techniques, we observed that on insulin-stimulated tissues, approximately 70% of phosphofructokinase activity is co-located in an actin-enriched fraction, against 28% in control. This phenomenon is accompanied by a 100% increase in specific phosphofructokinase activity in stimulated homogenates. Purified F-actin causes an increase of 230% in phosphofructokinase activity and alters its kinetic parameters. The presence of F-actin increases the affinity of phosphofructokinase for fructose 6-phosphate nevertheless, with no changes in maximum velocity (Vmax). Here we propose that the modulation of cellular distribution of phosphofructokinase may be one of the mechanisms of control of glycolytic flux in mammalian muscle by insulin.

Actins↗
PubMed1990

Activation of mammalian phosphofructokinases by ribose 1,5-bisphosphate.

Ribose 1,5-bisphosphate (Rib-1,5-P2), a newly discovered activator of rat brain phosphofructokinase, forms rapidly during the initiation of glycolytic flux and disappears within 20 s (Ogushi, S., Lawson, J.W. R., Dobson, G.P., Veech, R.L., and Uyeda, K. (1990) J. Biol. Chem. 265, 10943-10949). Activation of various mammalian phosphofructokinases and plant pyrophosphate-dependent phosphofructokinases by Rib-1,5-P2 was investigated. The order of decreasing potency for activation of rabbit muscle phosphofructokinase was: fructose (Fru) 2,6-P2, Rib-1,5-P2, Fru-1,6-P2, Glc-1,6-P2, phosphoribosylpyrophosphate, ribulose-1,5-P2, sedoheptulose-1,7-P2, and myoinositol-1,4-P2. The K0.5 values for activation by Rib-1,5-P2 of rat brain, rat liver, and rabbit muscle phosphofructokinases and potato and mung bean pyrophosphate-dependent phosphofructokinases were 64 nM, 230 nM, 82 nM, 710 nM, and 80 microM, respectively. The corresponding K0.5 values for Fru-2,6-P2 were 9, 8.6, 10, 7, and 65 nM, respectively. Rib-1,5-P2 was a competitive inhibitor of Fru-2,6-P2, binding to the muscle enzyme with Ki of 26 microM. Citrate increased the K0.5 for Rib-1,5-P2 without affecting the maximum activation, and AMP lowered the K0.5 for Rib-1,5-P2 without affecting the maximum activation. These effects of citrate and AMP were similar to those observed with Fru-2,6-P2 and different from those with Fru-1,6-P2. Rib-1,5-P2 is the second most potent activator of phosphofructokinase thus far discovered. The Rib-1,5-P2-activated conformation of the enzyme seems to be similar to that induced by Fru-2,6-P2, but different from that induced by Fru-1,6-P2.

Animals↗
PubMed1987

Regulation of adult and fetal myocardial phosphofructokinase. Relief of cooperativity and competition between fructose 2,6-bisphosphate, ATP, and citrate.

To clarify the physiological role of fructose 2,6-bisphosphate in the perinatal switching of myocardial fuels from carbohydrate to fatty acids, the kinetic effects of fructose 2,6-bisphosphate on phosphofructokinase purified from fetal and adult rat hearts were compared. For both enzymes at physiological pH and ATP concentrations, 1 microM fructose 2,6-bisphosphate induced a greater than 10-fold reduction in S0.5 for fructose 6-phosphate and it completely eliminated subunit cooperativity. Fructose 2,6-bisphosphate may thereby reduce the influence of changes in fructose 6-phosphate concentration on phosphofructokinase activity. Based on double-reciprocal plots and ATP inhibition studies, adult heart phosphofructokinase activity is more sensitive to physiological changes in ATP and citrate concentrations than to changes in fructose 2,6-bisphosphate concentrations. Fetal heart phosphofructokinase is less sensitive to ATP concentration above 5 mM and equally sensitive to citrate inhibition. The fetal enzyme has up to a 15-fold lower affinity for fructose 2,6-bisphosphate, rendering it more sensitive to changes in fructose 2,6-bisphosphate concentration than adult heart phosphofructokinase. Together, these factors allow greater phosphofructokinase activity in fetal heart while retaining sensitive metabolic control. In both fetal and adult heart, fructose 2,6-bisphosphate is primarily permissive: it abolishes subunit cooperativity and in its presence phosphofructokinase activity is extraordinarily sensitive to both the energy balance of the cell as reflected in ATP concentration and the availability of other fuels as reflected in cytosolic citrate concentration.

Adenosine Triphosphate↗
PubMed1985

Modulation of muscle phosphofructokinase at physiological concentration of enzyme.

Two approaches have been used to study the allosteric modulation of phosphofructokinase at physiological concentration of enzyme; a "slow motion" approach based on the use of a very low Mg2+/ATP ratio to conveniently lower Vmax, and the addition of polyethylene glycol as a "crowding" agent to favor aggregation of diluted enzyme. At 0.6 mg/ml muscle phosphofructokinase exhibited a drastic decrease in the ATP inhibition and the concomitant increase in the apparent affinity for fructose-6-P, as compared to a 100-fold diluted enzyme. Similar results were obtained with diluted enzyme in the presence of 10% polyethylene glycol (Mr = 6000). Results with these two approaches in vitro were essentially similar to those previously observed in situ (Aragón, J. J., Felíu, F. E., Frenkel, R., and Sols, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6324-6328), indicating that the enzyme is strongly dependent on homologous interactions at physiological concentrations. With polyethylene glycol it was observed that within the physiological range of concentration of substrates and the other positive effectors, fructose-2,6-P2 still activates the liver phosphofructokinase although it no longer significantly affects the muscle isozyme. In the presence of polyethylene glycol, muscle phosphofructokinase can approach its maximal rate even in the presence of physiologically high concentrations of ATP. Three minor activities of muscle phosphofructokinase have been studied at high enzyme concentration: the hydrolysis of MgATP (ATPase) and fructose-1,6-P2 (FBPase), produced in the absence of the other substrate, and the reverse reaction from MgADP and fructose-1,6-P2. The kinetic study of these activities has allowed a new insight into the mechanisms involved in the modulation of phosphofructokinase activity. The binding of (Mg)ATP at its regulatory site reduces the ability of the enzyme to cleave the bond of the terminal phosphate of MgATP at the substrate site. The positive effectors (Pi, cAMP, NH+4, fructose-1,6-P2, and fructose-2,6-P2) decrease the inhibitory effect of MgATP. Citrate and fructose-2,6-P2 both act as mechanistically "secondary" effectors in the sense that citrate does not inhibit and fructose-2,6-P2 does not activate the FBPase activity, requiring both the presence of ATP to affect the enzyme activity. In conclusion it appears that the regulatory behavior of mammalian phosphofructokinases is utterly dependent on the fact of their high concentrations in vivo.

Adenosine Triphosphatases↗
PubMed1985

AMP deaminase reaction as a control system of glycolysis in yeast. Role of ammonium ion in the interaction of phosphofructokinase and pyruvate kinase activity with the adenylate energy charge.

The role of ammonium ion and AMP deaminase (EC 3.5.4.6) reaction in the activation of phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) by the decrease in the adenylate energy charge was investigated using permeabilized yeast cells. Response of AMP deaminase, phosphofructokinase, and pyruvate kinase to variation in the energy charge is typical of the ATP-regenerating enzymes: an activation with the decrease in the energy charge under the in situ conditions. The addition of polyamine activated AMP deaminase in situ, resulting in the subsequent increase in ammonium production, which can stimulate the phosphofructokinase activity with the increase in the optimal energy charge value giving maximal activity of the enzyme. The optimal energy charge value of phosphofructokinase was 0.2-0.25 in the absence of ammonium ion and was shifted to the value above 0.5 by the addition of ammonium ion, whereas Pi, an activator of the enzyme showed little effect on the increase in the optimal energy charge value. The optimal energy charge value of AMP deaminase and pyruvate kinase was not affected by the addition of their effectors. Modulation of the response to the energy charge of phosphofructokinase and pyruvate kinase was analyzed in terms of the "activation coefficient," which was defined as the ratio of the activity at the energy charge of 0.6 to that at the value of 0.9. Activation of phosphofructokinase by the physiological decrease in the energy charge (0.9 to 0.6) can be enhanced by the increase in ammonium ion specifically, although the coefficient of pyruvate kinase remained unaffected by ammonium ion. These results suggest that the AMP deaminase reaction as an ammonium-forming reaction can participate in a key role in the stimulation of phosphofructokinase or glycolytic flux in cells.

AMP Deaminase↗
PubMed1965

PROPERTIES OF PHOSPHOFRUCTOKINASE FROM RAT LIVER AND THEIR RELATION TO THE CONTROL OF GLYCOLYSIS AND GLUCONEOGENESIS.

1. Phosphofructokinase from rat liver has been partially purified by ammonium sulphate precipitation so as to remove enzymes that interfere in one assay for phosphofructokinase. The properties of this enzyme were found to be similar to those of the same enzyme from other tissues (e.g. cardiac muscle, skeletal muscle and brain) that were previously investigated by other workers. 2. Low concentrations of ATP inhibited phosphofructokinase activity by decreasing the affinity of the enzyme for the other substrate, fructose 6-phosphate. Citrate, and other intermediates of the tricarboxylic acid cycle, also inhibited the activity of phosphofructokinase. 3. This inhibition was relieved by either AMP or fructose 1,6-diphosphate; however, higher concentrations of ATP decreased and finally removed the effect of these activators. 4. Ammonium sulphate protected the enzyme from inactivation, and increased the activity by relieving the inhibition due to ATP. The latter effect was similar to that of AMP. 5. Phosphofructokinase was found in the same cellular compartment as fructose 1,6-diphosphatase, namely the soluble cytoplasm. 6. The properties of phosphofructokinase and fructose 1,6-diphosphatase are compared and a theory is proposed that affords dual control of both enzymes in the liver. The relation of this to the control of glycolysis and gluconeogenesis is discussed.

Adenine Nucleotides↗
PubMed1957

The relationship between inhibition of phosphofructokinase activity and the mode of action of trivalent organic antimonials on Schistosoma mansoni.

The addition of purified mammalian phosphofructokinase to homogenates of schistosoma mansoni increased the rate of lactic acid production from glucose and reversed the inhibition of glycolysis produced by low concentrations of trivalent organic antimonials. Neither mammalian phosphofructokinase nor trivalent antimonials affected the rate of lactic acid production from fructose-1:6-diphosphate (HDP) by schistosome homogenates. Accordingly, in the schistosome, the rate of glycolysis of glucose is determined by the activity of phosphofructokinase.The aldolase of S. mansoni has a high requirement for HDP; relatively slight reductions in the concentration of this substrate below the optimum resulted in a sharp decline of aldolase activity. Therefore, decreased formation of HDP, due to inhibition of schistosome phosphofructokinase activity by antimonials, reduced the activity of aldolase and resulted in an inhibition of glycolysis of schistosome homogenates.Kinetic data revealed differences in the nature of the phosphofructokinase of S. mansoni and that of the enzyme catalysing the same reaction in the host. Exposure of schistosomes to low concentrations of potassium antimonyl tartrate or administration of subcurative doses of stibophen to the host resulted in an accumulation of the substrate (fructose-6-phosphate), and a reduction of the product (HDP) of the phosphofructokinase reaction, indicating that the activity of this enzyme was inhibited by antimonials in the intact parasite. It is concluded that inhibition of phosphofructokinase activity can account for the mechanism of the chemotherapeutic action of trivalent organic antimonials in schistosomiasis.

Animals↗