The Experts below are selected from a list of 19059 Experts worldwide ranked by ideXlab platform
Judit Ovadi - One of the best experts on this subject based on the ideXlab platform.
-
Quantitative characterization of homo- and heteroassociations of muscle Phosphofructokinase with aldolase.
Biochimica et biophysica acta, 2000Co-Authors: B. Rais, J Puigjaner, B Comin, Ferenc Orosz, F. Ortega, Judit Ovadi, Marta CascanteAbstract: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.
-
Specific characteristics of Phosphofructokinase-microtubule interaction.
FEBS Letters, 1996Co-Authors: Beáta G. Vértessy, János Kovács, Judit OvadiAbstract:Muscle Phosphofructokinase interacts with microtubule-associated protein-free microtubules resulting in a reduction of the overall activity of the enzyme [Lehotzky et al. (1993) J. Biol. Chem. 268,10888–10894] and periodical cross-linking of the tubules [Lehotzky et al. (1994) Biochem. Biophys. Res. Commun. 204, 585–5911. Microtubule polymers of ‘tail-free’ tubulin obtained by removal of the carboxy-termini with limited subtilisin digestion retain the binding domains for Phosphofructokinase that cross-bridges microtubule ‘bodies’. Microtubule-associated proteins bound on tubulin ‘tails’ do not perturb the kinase binding. These data suggest that the tubulin carboxy-terminal domain is not involved in microtubule-Phosphofructokinase interactions and Phosphofructokinase and microtubule-associated proteins have distinct binding domains on microtubules. Of different isoforms of Phosphofructokinase, occurring mainly in brain and tumor cells, the muscle isoform exhibits selective adsorption behaviour on microtubules. Phosphofructokinase M and C isoforms with different associative and allosteric properties may represent an auxiliary pathway to modulate energy production via glycolysis.
-
Specific characteristics of Phosphofructokinase‐microtubule interaction
FEBS letters, 1996Co-Authors: Beáta G. Vértessy, János Kovács, Judit OvadiAbstract:Muscle Phosphofructokinase interacts with microtubule-associated protein-free microtubules resulting in a reduction of the overall activity of the enzyme [Lehotzky et al. (1993) J. Biol. Chem. 268,10888–10894] and periodical cross-linking of the tubules [Lehotzky et al. (1994) Biochem. Biophys. Res. Commun. 204, 585–5911. Microtubule polymers of ‘tail-free’ tubulin obtained by removal of the carboxy-termini with limited subtilisin digestion retain the binding domains for Phosphofructokinase that cross-bridges microtubule ‘bodies’. Microtubule-associated proteins bound on tubulin ‘tails’ do not perturb the kinase binding. These data suggest that the tubulin carboxy-terminal domain is not involved in microtubule-Phosphofructokinase interactions and Phosphofructokinase and microtubule-associated proteins have distinct binding domains on microtubules. Of different isoforms of Phosphofructokinase, occurring mainly in brain and tumor cells, the muscle isoform exhibits selective adsorption behaviour on microtubules. Phosphofructokinase M and C isoforms with different associative and allosteric properties may represent an auxiliary pathway to modulate energy production via glycolysis.
-
Interaction of Phosphofructokinase with tubulin and microtubules. Quantitative evaluation of the mutual effects.
The Journal of biological chemistry, 1993Co-Authors: Attila Lehotzky, Marianna Telegdi, Károly Liliom, Judit OvadiAbstract:Abstract 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.
Hugh W. Morgan - One of the best experts on this subject based on the ideXlab platform.
-
the biochemical properties and phylogenies of Phosphofructokinases from extremophiles
Extremophiles, 2001Co-Authors: Ron S. Ronimus, Hugh W. MorganAbstract:The enzyme Phosphofructokinase (PFK) is a defining activity of the highly conserved glycolytic pathway, and is present in the domains Bacteria, Eukarya, and Archaea. PFK subtypes are now known that utilize either ATP, ADP, or pyrophosphate as the primary phosphoryl donor and share the ability to catalyze the transfer of phosphate to the 1-position of fructose-6-phosphate. Because of the crucial position in the glycolytic pathway of PFKs, their biochemical characteristics and phylogenies may play a significant role in elucidating the origins of glycolysis and, indeed, of metabolism itself. Despite the shared ability to phosphorylate fructose-6-phosphate, PFKs that have been characterized to date now fall into three sequence families: the PFKA family, consisting of the well-known higher eukaryotic ATP-dependent PFKs together with their ATP- and pyrophosphate-dependent bacterial cousins (including the crenarchaeal pyrophosphate-dependent PFK of Thermoproteus tenax) and plant pyrophosphate-dependent Phosphofructokinases; the PFKB family, exemplified by the minor ATP-dependent PFK activity of Escherichia coli (PFK 2), but which also includes at least one crenarchaeal enzyme in Aeropyrum pernix; and the tentatively named PFKC family, which contains the unique ADP-dependent PFKs from the euryarchaeal genera of Pyrococcus and Thermococcus, which are indicated by sequence analysis to be present also in the methanogenic species Methanococcus jannaschii and Methanosarcina mazei.
-
Sequencing, high-level expression and phylogeny of the pyrophosphate-dependent Phosphofructokinase from the thermophilic spirochete Spirochaeta thermophila.
Archives of Microbiology, 2001Co-Authors: Ron S. Ronimus, Edwin De Heus, Andreas Rückert, Hugh W. MorganAbstract:The full-length gene encoding a 554-amino-acid, active pyrophosphate-dependent Phosphofructokinase from Spirochaeta thermophila was cloned and sequenced using a combination of degenerate and inverse PCR, and the enzyme expressed to a high level in Escherichia coli. The recombinant enzyme, with a calculated molecular mass of 61 kDa, was purified to near homogeneity and found to be similar to the purified native enzyme for most properties examined. Phylogenetic analysis demonstrated a close relationship between the thermophilic S. thermophila Phosphofructokinase and the large β-subunits of the Phosphofructokinases from Borrelia burgdorferi and Treponema pallidum.
-
Phosphofructokinase activities within the order Spirochaetales and the characterisation of the pyrophosphate-dependent Phosphofructokinase from Spirochaeta thermophila
Archives of Microbiology, 1999Co-Authors: Ron S. Ronimus, Hugh W. Morgan, Yan-huai Richard DingAbstract:The subtype of Phosphofructokinase activity, either ATP-, ADP- or pyrophosphate-dependent, present in members of three genera from the Spirochaetales was investigated. The individual species/strains examined included Spirochaeta alkalica, S. asiatica, S. halophila, S. isovalerica, S. litoralis, S. zuelzerae, S. thermophila, two thermophilic spirochetes, Treponema bryantii, T. denticola, ¶T. pectinovorum, Leptospira biflexa and L. interrogans. All of the Spirochaeta strains, regardless of their phenotype, possessed primarily a pyrophosphate-dependent Phosphofructokinase. In contrast, T. bryantii, T. denticola and L. biflexa had predominantly an ATP-dependent activity, whereas no activity was detected in T. pectinovorum or ¶L. interrogans. The results suggest that pyrophosphate-dependent Phosphofructokinase activity may be a reliable phenotypic marker for the genus Spirochaeta and that there are potentially interesting differences in how the catabolism of saccharides is controlled among members of genera within the Spirochaetales. The pyrophosphate-dependent Phosphofructokinase from S. thermophila strain RI 19.B1 was purified (303-fold) to homogeneity and biochemically characterised. The S. thermophila enzyme displayed hyperbolic kinetics with respect to both the forward and reverse cosubstrates and was not significantly affected by traditional activators or inhibitors of Phosphofructokinase. The biochemical characterisation represents the first spirochete Phosphofructokinase to be described.
Mauro Sola-penna - One of the best experts on this subject based on the ideXlab platform.
-
Effects of insulin and actin on Phosphofructokinase activity and cellular distribution in skeletal muscle
Anais Da Academia Brasileira De Ciencias, 2004Co-Authors: Ana Paula Pereira Da Silva, Gutemberg Gomes Alves, Alexandre Herculano Borges De Araújo, Mauro Sola-pennaAbstract: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.
-
Epinephrine modulates cellular distribution of muscle Phosphofructokinase
Molecular Genetics and Metabolism, 2003Co-Authors: Gutemberg Gomes Alves, Mauro Sola-pennaAbstract:In this paper, we report evidences that cellular distribution of Phosphofructokinase can be affected by epinephrine stimulation in rabbit skeletal muscle homogenates. Through co-sedimentation techniques, we observed that in epinephrine-stimulated tissues, approximately 50% of Phosphofructokinase activity is co-located in an actin-enriched fraction, against 29% in control. This phenomenon is companied by a 400% increase in specific Phosphofructokinase activity in stimulated homogenates. This effect is reproduced by the β-adrenergic agonist isoprenaline. 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 β-adrenergic stimulation.
Ron S. Ronimus - One of the best experts on this subject based on the ideXlab platform.
-
the biochemical properties and phylogenies of Phosphofructokinases from extremophiles
Extremophiles, 2001Co-Authors: Ron S. Ronimus, Hugh W. MorganAbstract:The enzyme Phosphofructokinase (PFK) is a defining activity of the highly conserved glycolytic pathway, and is present in the domains Bacteria, Eukarya, and Archaea. PFK subtypes are now known that utilize either ATP, ADP, or pyrophosphate as the primary phosphoryl donor and share the ability to catalyze the transfer of phosphate to the 1-position of fructose-6-phosphate. Because of the crucial position in the glycolytic pathway of PFKs, their biochemical characteristics and phylogenies may play a significant role in elucidating the origins of glycolysis and, indeed, of metabolism itself. Despite the shared ability to phosphorylate fructose-6-phosphate, PFKs that have been characterized to date now fall into three sequence families: the PFKA family, consisting of the well-known higher eukaryotic ATP-dependent PFKs together with their ATP- and pyrophosphate-dependent bacterial cousins (including the crenarchaeal pyrophosphate-dependent PFK of Thermoproteus tenax) and plant pyrophosphate-dependent Phosphofructokinases; the PFKB family, exemplified by the minor ATP-dependent PFK activity of Escherichia coli (PFK 2), but which also includes at least one crenarchaeal enzyme in Aeropyrum pernix; and the tentatively named PFKC family, which contains the unique ADP-dependent PFKs from the euryarchaeal genera of Pyrococcus and Thermococcus, which are indicated by sequence analysis to be present also in the methanogenic species Methanococcus jannaschii and Methanosarcina mazei.
-
Sequencing, high-level expression and phylogeny of the pyrophosphate-dependent Phosphofructokinase from the thermophilic spirochete Spirochaeta thermophila.
Archives of Microbiology, 2001Co-Authors: Ron S. Ronimus, Edwin De Heus, Andreas Rückert, Hugh W. MorganAbstract:The full-length gene encoding a 554-amino-acid, active pyrophosphate-dependent Phosphofructokinase from Spirochaeta thermophila was cloned and sequenced using a combination of degenerate and inverse PCR, and the enzyme expressed to a high level in Escherichia coli. The recombinant enzyme, with a calculated molecular mass of 61 kDa, was purified to near homogeneity and found to be similar to the purified native enzyme for most properties examined. Phylogenetic analysis demonstrated a close relationship between the thermophilic S. thermophila Phosphofructokinase and the large β-subunits of the Phosphofructokinases from Borrelia burgdorferi and Treponema pallidum.
-
Phosphofructokinase activities within the order Spirochaetales and the characterisation of the pyrophosphate-dependent Phosphofructokinase from Spirochaeta thermophila
Archives of Microbiology, 1999Co-Authors: Ron S. Ronimus, Hugh W. Morgan, Yan-huai Richard DingAbstract:The subtype of Phosphofructokinase activity, either ATP-, ADP- or pyrophosphate-dependent, present in members of three genera from the Spirochaetales was investigated. The individual species/strains examined included Spirochaeta alkalica, S. asiatica, S. halophila, S. isovalerica, S. litoralis, S. zuelzerae, S. thermophila, two thermophilic spirochetes, Treponema bryantii, T. denticola, ¶T. pectinovorum, Leptospira biflexa and L. interrogans. All of the Spirochaeta strains, regardless of their phenotype, possessed primarily a pyrophosphate-dependent Phosphofructokinase. In contrast, T. bryantii, T. denticola and L. biflexa had predominantly an ATP-dependent activity, whereas no activity was detected in T. pectinovorum or ¶L. interrogans. The results suggest that pyrophosphate-dependent Phosphofructokinase activity may be a reliable phenotypic marker for the genus Spirochaeta and that there are potentially interesting differences in how the catabolism of saccharides is controlled among members of genera within the Spirochaetales. The pyrophosphate-dependent Phosphofructokinase from S. thermophila strain RI 19.B1 was purified (303-fold) to homogeneity and biochemically characterised. The S. thermophila enzyme displayed hyperbolic kinetics with respect to both the forward and reverse cosubstrates and was not significantly affected by traditional activators or inhibitors of Phosphofructokinase. The biochemical characterisation represents the first spirochete Phosphofructokinase to be described.
Jorge Babul - One of the best experts on this subject based on the ideXlab platform.
-
crystallographic structure of Phosphofructokinase 2 from escherichia coli in complex with two atp molecules implications for substrate inhibition
Journal of Molecular Biology, 2008Co-Authors: Ricardo Cabrera, Victoria Guixe, Andre Luis Berteli Ambrosio, Richard Charles Garratt, Jorge BabulAbstract:Phosphofructokinase-1 and -2 (Pfk-1 and Pfk-2, respectively) from Escherichia coli belong to different homologous superfamilies. However, in spite of the lack of a common ancestor, they share the ability to catalyze the same reaction and are inhibited by the substrate MgATP. Pfk-2, an ATPdependent 6-Phosphofructokinase member of the ribokinase-like superfamily, is a homodimer of 66 kDa subunits whose oligomerization state is necessary for catalysis and stability. The presence of MgATP favors the tetrameric form of the enzyme. In this work, we describe the structure of Pfk-2 in its inhibited tetrameric form, with each subunit bound to two ATP molecules and two Mg ions. The present structure indicates that substrate inhibition occurs due to the sequential binding of two MgATP molecules per subunit, the first at the usual site occupied by the nucleotide in homologous enzymes and the second at the allosteric site, making a number of direct and Mg-mediated interactions with the first. Two configurations are observed for the second MgATP, one of which involves interactions with Tyr23 from the adjacent subunit in the dimer and the other making an unusual nonWatson-Crick base pairing with the adenine in the substrate ATP. The oligomeric state observed in the crystal is tetrameric, and some of the structural elements involved in the binding of the substrate and allosteric ATPs are also participating in the dimer–dimer interface. This structure also provides the grounds to compare analogous features of the nonhomologous Phosphofructokinases from E. coli.
-
an alteration in Phosphofructokinase 2 of escherichia coli which impairs gluconeogenic growth and improves growth on sugars
FEBS Journal, 2005Co-Authors: Fevzi Daldal, Jorge Babul, Victoria Guixe, Dan G. FraenkelAbstract:Escherichia coli contains a major Phosphofructokinase isoenzyme, Phosphofructokinase 1, which is allosteric, and a minor isoenzyme, Phosphofructokinase 2. The pfkBl mutation is known to increase the amount of Phosphofructokinase 2 and allow growth on sugars of mutants lacking Phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., ‘gluconeogenic growth’). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkBl*. We show here that strains with pfkBl* contain an altered form of Phosphofructokinase 2, called Phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from Phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6-bisphosphate. The Km for fructose 6-phosphate is low (about 5 × 10−5 M) in both Phosphofructokinase 2 and Phosphofructokinase 2*. However, in strains lacking Phosphofructokinase 1, a high level of Phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with Phosphofructokinase 2* instead of Phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in Phosphofructokinase 2 strains, while the impaired growth of Phosphofructokinase 2* strains is associated with high levels of fructose 2,6-bisphosphate and very low levels of hexose monophosphates. These results show that Phosphofructokinase 2, as studied in vitro, should no longer be regarded as a ‘non-allosteric’ protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569–574 (1981)]. The fact that mutational alteration of Phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6-bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.
-
A mutant Phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli.
Biochemical Journal, 1997Co-Authors: C. Juan Torres, Victoria Guixe, Jorge BabulAbstract:Strains of Escherichia coli bearing different forms of Phosphofructokinase were used to assess the occurrence of futile cycling in cell resuspensions supplied with glycerol as gluconeogenic carbon source. A model was used to simulate results of different kinds of experiments for different levels of futile cycle. The main predictions of the model were experimentally confirmed in a strain with a mutant Phosphofructokinase-2 (Phosphofructokinase-2*) which is not inhibited by MgATP. The intracellular fructose 1, 6-bisphosphate concentration reaches significantly higher levels in the mutant-bearing strain than in strains with either Phosphofructokinase-1 or -2. Also, this strain showed a higher rate and level of in vivo radioactive labelling of fructose 1, 6-bisphosphate, from a trace of [U-14C]glucose supplied during gluconeogenesis, indicating higher kinase activity in these conditions. Cell resuspensions of the mutant-bearing strain produced higher levels of radioactively labelled CO2 when supplied with [U-14C]glycerol as the only carbon source. Simultaneously, fewer glycerol carbons were incorporated into HClO4-insoluble macromolecules. Finally, radioactive CO2 output was measured in resuspensions supplied with glycerol as the major carbon source with traces of either [1-14C]glucose or [6-14C]glucose. It was found that, whereas in the strains with either of the wild-type Phosphofructokinase isoenzymes, radioactive CO2 output from [1-14C]glucose was higher than with [6-14C]glucose, the reverse is found for the strain with Phosphofructokinase-2*. This result also agrees with the corresponding prediction of the model. Using the radioactivity flux rates predicted by the model, an explanation linking the futile cycle to the differential labelling of CO2 is advanced. Finally, on the basis of these results it is proposed that strains bearing Phosphofructokinase-2* sustain higher rates of futile cycling during gluconeogenesis than strains bearing either of the wild-type isoforms of Phosphofructokinase. The kinetic equations and parameter values used for the model simulations are given in Supplementary Publication SUP 50183 (8 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8.
-
an in vitro model showing different rates of substrate cycle for Phosphofructokinases of escherichia coli with different kinetic properties
FEBS Journal, 1991Co-Authors: Juan Carlos Torres, Jorge BabulAbstract:An in vitro assay model is introduced for the coupled assay of Phosphofructokinase (PFK) and fructosebisphosphatase. The model is applied to the study of three PFK of Escherichia coli: two isoenzymes, Phosphofructokinase-1 (PFK-1) and Phosphofructokinase-2 (PFK-2), and a mutant form of Phosphofructokinase- 2 (PFK-2*). Results show that for a variety of conditions the PFK-1/fructose-bisphosphatase pair gives the lowest and the PFK-2*/fructose-bisphosphatase pair the highest rates of substrate cycle, with the PFK-2/fructose-bisphosphatase pair in an intermediate position. The effects of variables such as maximum activity ratios and MgATP concentration were explored. The possible role of MgATP in decreasing the futile cycle of the PFK-2/fructose-bisphosphatase pair is described. The results are discussed in terms of possible metabolic consequences of PFK-2* and of predictions of the model to be tested in vivo.
