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IUBMB Life Nov 2010Phosphofructokinase (PFK) is a major regulatory glycolytic enzyme and is considered to be the pacemaker of glycolysis. This enzyme presents a puzzling regulatory... (Review)
Review
Phosphofructokinase (PFK) is a major regulatory glycolytic enzyme and is considered to be the pacemaker of glycolysis. This enzyme presents a puzzling regulatory mechanism that is modulated by a large variety of metabolites, drugs, and intracellular proteins. To date, the mammalian enzyme structure has not yet been resolved. However, it is known that PFK undergoes an intricate oligomerization process, shifting among monomers, dimers, tetramers, and more complex oligomeric structures. The equilibrium between PFK dimers and tetramers is directly correlated with the enzyme regulation, because the dimer exhibits very low catalytic activity, whereas the tetramer is fully active. Several PFK ligands modulate the enzyme, favoring the formation of its dimers or tetramers. The present review integrates recent findings regarding the regulatory aspects of muscle type PFK and discusses their relation to the control of metabolism.
Topics: Actins; Allosteric Regulation; Animals; Anion Exchange Protein 1, Erythrocyte; Calcium; Calmodulin; Fructosediphosphates; Muscle, Skeletal; Phosphofructokinase-1, Muscle Type; Phosphorylation; Protein Multimerization; Protein Structure, Quaternary
PubMed: 21117169
DOI: 10.1002/iub.393 -
Proceedings of the National Academy of... Jun 1997The rules that govern the relationships between enzymatic flux capacities (Vmax) and maximum physiological flux rates (v) at enzyme-catalyzed steps in pathways are... (Review)
Review
The rules that govern the relationships between enzymatic flux capacities (Vmax) and maximum physiological flux rates (v) at enzyme-catalyzed steps in pathways are poorly understood. We relate in vitro Vmax values with in vivo flux rates for glycogen phosphorylase, hexokinase, and phosphofructokinase, enzymes catalyzing nonequilibrium reactions, from a variety of muscle types in fishes, insects, birds, and mammals. Flux capacities are in large excess over physiological flux rates in low-flux muscles, resulting in low fractional velocities (%Vmax = v/Vmax x 100) in vivo. In high-flux muscles, close matches between flux capacities and flux rates (resulting in fractional velocities approaching 100% in vivo) are observed. These empirical observations are reconciled with current concepts concerning enzyme function and regulation. We suggest that in high-flux muscles, close matches between enzymatic flux capacities and metabolic flux rates (i.e., the lack of excess capacities) may result from space constraints in the sarcoplasm.
Topics: Animals; Catalysis; Enzymes; Fishes; Glycolysis; Hexokinase; Kinetics; Muscles; Phosphofructokinase-1; Phosphorylases
PubMed: 9192692
DOI: 10.1073/pnas.94.13.7065 -
Analytical Biochemistry Feb 2014An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step...
An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg-ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg-ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by ultraviolet (UV) absorbance at 260 nm of Mg-ATP and Mg-ADP. The separation was enhanced by the addition of Mg²⁺ to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC₅₀ value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Electrophoresis, Capillary; Enzyme Assays; Enzyme Inhibitors; Phosphofructokinase-1; Rabbits
PubMed: 24444856
DOI: 10.1016/j.ab.2013.10.028 -
European Journal of Biochemistry Aug 1988Phosphofructokinase and gelsolin-like proteins coexist in many muscle and non-muscle tissues. They are both actin-binding proteins, and some of their biochemical...
Phosphofructokinase and gelsolin-like proteins coexist in many muscle and non-muscle tissues. They are both actin-binding proteins, and some of their biochemical parameters are remarkably similar. In a previous report [Füchtbauer, A., Jockusch, B. M., Leberer, E. & Pette, D. (1986) Proc. Natl Acad. Sci. USA 83, 9502-9506] it was shown that phosphofructokinase preparations contained actin-filament-severin activities characteristic for gelsolin. Therefore, we investigated a possible relationship between these proteins with respect to their actin-binding properties. Immunoblotting experiments with specific and non-cross-reacting antibodies to both proteins revealed two distinct polypeptides with slightly different molecular mass in SDS-PAGE of crude extracts from rabbit skeletal muscle, indicating that phosphofructokinase and gelsolin are not identical. An actin-filament-severing activity as well as the component detected by anti-gelsolin were found to copurify with phosphofructokinase during its preparation. However, the presumptive gelsolin was completely eliminated after a heat-denaturation step leaving the phosphofructokinase activity unaffected. Purified phosphofructokinase had no effects on the polymer state of preformed actin filaments. Unlike gelsolin, phosphofructokinase did not promote nucleation of actin polymerization but delayed the nucleation step. We therefore conclude that phosphofructokinase and gelsolin are functionally and structurally distinct proteins.
Topics: Actins; Animals; Antibodies; Antigen-Antibody Complex; Calcium-Binding Proteins; Cross Reactions; Gastric Mucosa; Gelsolin; Kinetics; Microfilament Proteins; Muscle, Smooth; Muscles; Phosphofructokinase-1; Protein Binding; Rabbits; Swine
PubMed: 2841131
DOI: 10.1111/j.1432-1033.1988.tb14190.x -
The Journal of Cell Biology Aug 2017Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell.... (Comparative Study)
Comparative Study
Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that nonglycolytic metabolic enzymes regulating diverse pathways can assemble into polymers. We now show tetramer- and substrate-dependent filament assembly by phosphofructokinase-1 (PFK1), which is considered the "gatekeeper" of glycolysis because it catalyzes the step committing glucose to breakdown. Recombinant liver PFK1 (PFKL) isoform, but not platelet PFK1 (PFKP) or muscle PFK1 (PFKM) isoforms, assembles into filaments. Negative-stain electron micrographs reveal that filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer. Electron micrographs and biochemical data with a PFKL/PFKP chimera indicate that the PFKL regulatory domain mediates filament assembly. Quantified live-cell imaging shows dynamic properties of localized PFKL puncta that are enriched at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform-specific glucose metabolism in cells.
Topics: Blood Platelets; Cell Membrane; Glucose; Glycolysis; HEK293 Cells; Humans; Kinetics; Liver; Microscopy, Confocal; Microscopy, Electron, Transmission; Microscopy, Video; Muscle, Skeletal; Phosphofructokinase-1, Liver Type; Phosphofructokinase-1, Muscle Type; Phosphofructokinase-1, Type C; Protein Multimerization; Protein Structure, Quaternary; Recombinant Proteins; Structure-Activity Relationship; Substrate Specificity; Time-Lapse Imaging
PubMed: 28646105
DOI: 10.1083/jcb.201701084 -
The Journal of Veterinary Medical... May 2019Phosphofructokinase-1 (EC:2.7.1.11, PFK-1) catalyzes the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate using adenosine triphosphate and is a key...
Phosphofructokinase-1 (EC:2.7.1.11, PFK-1) catalyzes the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate using adenosine triphosphate and is a key regulatory enzyme of glycolysis. Mammalian PFK-1 isozymes are composed of three kinds of subunits (PFK-M, -L, and -P), with different properties. It has been suggested that the proportion of PFK-1 subunits in different organs is based on the organ energy metabolism. In this study, we analyzed the activity and subunit composition of canine PFK-1. We found that, in dogs, the skeletal muscle only has PFK-M, the liver mainly has PFK-L, and the brain expresses all of them. The knowledge of the composition of PFK-1 could provide useful information for determination of the differences in glycolysis in various organs of dogs.
Topics: Animals; Brain; Dogs; Female; Isoenzymes; Liver; Male; Muscle, Skeletal; Phosphofructokinase-1; Tissue Distribution
PubMed: 30918224
DOI: 10.1292/jvms.19-0049 -
The Biochemical Journal Jun 19651. Phosphofructokinase from rat liver has been partially purified by ammonium sulphate precipitation so as to remove enzymes that interfere in one assay for...
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.
Topics: Adenine Nucleotides; Adenosine Triphosphate; Carbohydrate Metabolism; Citrates; Enzyme Inhibitors; Fructose; Gluconeogenesis; Glucose; Glycolysis; Hexosephosphates; Liver; Pharmacology; Phosphofructokinase-1; Phosphofructokinases; Rats; Research
PubMed: 14342527
DOI: 10.1042/bj0950868 -
The Biochemical Journal Dec 1984A newly developed specific radioimmunoassay was used to quantify phosphofructokinase protein directly and independently of assayable activity in liver and kidney cytosol...
A newly developed specific radioimmunoassay was used to quantify phosphofructokinase protein directly and independently of assayable activity in liver and kidney cytosol of normal fed, starved and alloxan-diabetic rats. In the fed state, liver phosphofructokinase concentration was 0.096 microM and the kidney enzyme was 0.086 microM (mumol/kg of tissue). In the starved state (24h), liver and kidney phosphofructokinase concentrations decreased by 30%. Prolonged starvation up to 72h did not further decrease enzyme concentration. In liver, total enzyme content during starvation declined by more than 50%, secondary also to a decrease in liver weight. In the alloxan-diabetic rats, there was a 22% decrease in enzyme protein concentration in liver and kidney. Total enzyme content per liver actually decreased much more (46%), because diabetes also resulted in a decrease in liver size. In conjunction with assayable activity measurements, the results of the radioimmunoassay allowed us to calculate the apparent specific activity of the enzyme. The specific activity of the kidney enzyme was 2-3 times that of the liver. Little or no change in specific activity of the liver or kidney enzyme occurred as a result of starvation or chemically induced diabetes. Tissue enzyme concentrations of phosphofructokinase unequivocally reconcile the ultimate results of changing rates of synthesis and degradation and are useful data in the design of spectrophotometric, kinetic, aggregation-disaggregation and other studies.
Topics: Animals; Diabetes Mellitus, Experimental; Food; Half-Life; Immune Sera; Kidney; Liver; Male; Phosphofructokinase-1; Radioimmunoassay; Rats; Rats, Inbred Strains; Starvation
PubMed: 6240262
DOI: 10.1042/bj2240541 -
The Journal of Biological Chemistry May 1993The 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...
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.
Topics: Animals; Brain; Cattle; Electrophoresis, Polyacrylamide Gel; Fluorescein-5-isothiocyanate; Fluorescence Polarization; Kinetics; Macromolecular Substances; Mathematics; Microtubules; Models, Biological; Paclitaxel; Phosphofructokinase-1; Serum Albumin, Bovine; Tubulin
PubMed: 8098705
DOI: No ID Found -
The Journal of Biological Chemistry Aug 1981Phosphofructokinase isozymes of fetal, neonatal, and adult rat heart and skeletal muscle were characterized by DEAE-cellulose chromatography, agarose gel...
Phosphofructokinase isozymes of fetal, neonatal, and adult rat heart and skeletal muscle were characterized by DEAE-cellulose chromatography, agarose gel electrophoresis, and immunodiffusion with specific antisera. The results of these studies indicate that in skeletal muscle and heart the levels of the major liver phosphofructokinase isozyme (PFK-L2) and the muscle phosphofructokinase isozyme (PFK-M) are dependent on the developmental status of the rat. For example, PFK-L2 and PFK-M are present in fetal and early neonatal skeletal muscle; whereas in adult skeletal muscle, only PFK-M is detectable. By DEAE- cellulose chromatography, PFK-L2 activity was estimated to be 2.4 units/g (41% of total phosphofructokinase activity) in fetal muscle, very low and not resolved from PFK-M in 7-day neonatal muscle, and not detectable in adult muscle. Further, PFK-M activity was found to be 3.4 units/g (59% of total phosphofructokinase activity), 10 units/g, and 31.6 units/g in fetal, 7-day neonatal, and adult skeletal muscle, respectively. The developmental changes of heart phosphofructokinase isozymes differ considerably from that of the skeletal muscle phosphofructokinase isozymes. In fetal heart, PFK-L2 is the major phosphofructokinase isozyme (5.6 units/g), constituting 67% of total phosphofructokinase activity. Further, in fetal heart another phosphofructokinase isozyme (33% of total phosphofructokinase activity) was found by DEAE-cellulose chromatography which is different from PFK-M and PFK-L2. In 7-day neonatal and adult heart, PFK-M and PFK-L2 are the only detectable phosphofructokinase isozymes. Varying from 5.6 units/g (44% of total) in 7-day neonatal to 5.9 units/g (40% of total) in adult heart, PFK-L2 activity remains fairly constant. Also, PFK-M is very low in fetal heart but increases within 1 week postpartum to 5.5 units/g (50% of total activity) and to 8.9 units/g (60% of total activity) in adult heart.
Topics: Aging; Animals; Animals, Newborn; Fetus; Heart; Immunodiffusion; Isoenzymes; Muscle Development; Muscles; Myocardium; Phosphofructokinase-1; Rats
PubMed: 6455419
DOI: No ID Found