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Journal of Molecular Biology Jan 1968
Topics: Adenosine Triphosphate; Escherichia coli; Hexosephosphates; Kinetics; Nucleotides; Phosphofructokinase-1; Pyruvates
PubMed: 4229913
DOI: 10.1016/0022-2836(68)90051-x -
Journal of Chromatography Nov 1988A novel purification of phosphofructokinase has been achieved in a two step process using ion-exchange affinity chromatography and a transition-state analogue affinity...
A novel purification of phosphofructokinase has been achieved in a two step process using ion-exchange affinity chromatography and a transition-state analogue affinity column matrix. The procedure can be performed in one day, and gives a 25% yield of the starting material. The transition-state analogue chromatography is carried out using an ADP-agarose column in the presence of fructose 6-phosphate, magnesium ions and nitrate ions. In the presence of nitrate ion plus substrate, phosphofructokinase binds immobilized ADP while other proteins pass through the column. Previous studies with creatine kinase have shown that the nitrate ion mimics the planar phosphate in the transition state resulting in a complex which is stable under the relatively high ionic strength of the column buffer. This permits the elution of phosphofructokinase in a single peak of high specific activity. This column typically results in a 20-30 fold increase in specific activity with only a small loss of activity.
Topics: Animals; Chromatography, Affinity; Phosphofructokinase-1; Turtles
PubMed: 2976771
DOI: 10.1016/s0021-9673(01)82127-0 -
Trends in Biochemical Sciences Dec 1994
Topics: Glycolysis; Homeostasis; Metabolism; Phosphofructokinase-1
PubMed: 7846757
DOI: 10.1016/0968-0004(94)90048-5 -
Biochemistry and Molecular Biology... Sep 1997Phosphofructokinase from mantle tissue of the sea mussel Mytilus galloprovincialis was phosphorylated in vitro by a protein kinase isolated from the same tissue,...
Phosphofructokinase from mantle tissue of the sea mussel Mytilus galloprovincialis was phosphorylated in vitro by a protein kinase isolated from the same tissue, homologous to mammalian cAMP-dependent protein kinase; the maximal level of phosphorylation achieved was around 1 mol of Pi/mol of phosphofructokinase subunit. The covalent incorporation of phosphate leads to a notable increase in the enzyme activity assayed at near-physiological concentrations of substrates and allosteric modulators and neutral pH. Tryptic digestion of labeled phosphofructokinase released a phosphopeptide whose sequence was Lys-Asp-Ser(P)-Ile-Trp-Ile-Gln-Thr-Gly-Arg. This sequence showed high homology with the phosphopeptides from other invertebrates whose phosphofructokinase is also activated by cAMP-dependent phosphorylation.
Topics: Amino Acid Sequence; Animals; Bivalvia; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Hydrogen-Ion Concentration; Phosphofructokinase-1; Phosphopeptides; Phosphorylation
PubMed: 9315295
DOI: 10.1080/15216549700203941 -
The Biochemical Journal Nov 1975Pig spleen phosphofructokinase has been purified 800-fold with a yield of 17%. Two isoenzymes that appear to be kinetically identical can be separated by DEAE-cellulose...
Pig spleen phosphofructokinase has been purified 800-fold with a yield of 17%. Two isoenzymes that appear to be kinetically identical can be separated by DEAE-cellulose column chromatography. In common with the enzyme from other mammalian sources, the spleen enzyme has a pH optimum of 8.2. At pH 7.0 it displays sigmoidal kinetics with respect to fructose 6-phosphate concentration but its co-operative behaviour is very dependent on pH, protein concentration and the concentration of MgATP. MgGTP and MgITP can replace MgATP as phosphate donors but, unlike MgATP, these nucleotides do not cause significant inhibition. Mn2+ and Co2+ (as the metal ion-ATP complexes) act as cofactors and in the free form are far more inhibitory than free Mg2+. The spleen enzyme responds to a wide variety of potential effector molecules: ADP, AMP, cyclic AMP, aspartate, NH4+, fructose 6-phosphate, fructose 1,6-diphosphate and Pi all act as either activators or protectors, whereas Mg-ATP, Mg2+, citrate, phosphoenol-pyruvate and the phosphoglucerates are inhibitors.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Ammonia; Animals; Chromatography, DEAE-Cellulose; Cyclic AMP; Enzyme Activation; Glycerophosphates; Hydrogen-Ion Concentration; Isoenzymes; Kinetics; Magnesium; Phosphoenolpyruvate; Phosphofructokinase-1; Potassium; Rubidium; Spleen; Swine
PubMed: 3166
DOI: 10.1042/bj1510327 -
Acta Crystallographica. Section F,... May 2014Whereas the three-dimensional structure and the structural basis of the allosteric regulation of prokaryotic 6-phosphofructokinases (Pfks) have been studied in great...
Whereas the three-dimensional structure and the structural basis of the allosteric regulation of prokaryotic 6-phosphofructokinases (Pfks) have been studied in great detail, knowledge of the molecular basis of the allosteric behaviour of the far more complex mammalian Pfks is still very limited. The human muscle isozyme was expressed heterologously in yeast cells and purified using a five-step purification protocol. Protein crystals suitable for diffraction experiments were obtained by the vapour-diffusion method. The crystals belonged to space group P6222 and diffracted to 6.0 Å resolution. The 3.2 Å resolution structure of rabbit muscle Pfk (rmPfk) was placed into the asymmetric unit and optimized by rigid-body and group B-factor refinement. Interestingly, the tetrameric enzyme dissociated into a dimer, similar to the situation observed in the structure of rmPfk.
Topics: Amino Acid Sequence; Crystallization; Crystallography; Glycolysis; Humans; Molecular Sequence Data; Muscle, Skeletal; Phosphofructokinase-1, Muscle Type; Protein Structure, Secondary; Protein Structure, Tertiary
PubMed: 24817713
DOI: 10.1107/S2053230X14008723 -
Yeast (Chichester, England) Mar 1998Phosphofructokinase-1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non-identical subunits, alpha and beta, which are encoded by the unlinked genes...
Phosphofructokinase-1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non-identical subunits, alpha and beta, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly and reactivation of the enzyme have been studied in cell-free extracts of single-deletion mutants. In contrast to the previously described lack of phosphofructokinase-1 activity in cell-free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts. This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption. Upon mixing of separately prepared cell-free extracts of both deletion mutants very low activity could be measured. About 40% of the wild-type activity was regained when both mutants were mixed prior to disruption. The reactivation rate could be slightly increased by addition of ATP and fructose 6-phosphate and was found to be a function of the growth state, particularly of the beta-subunit-carrying cells. The individual subunits did not interact with Cibacron Blue F3G-A, a biomimetic ligand of phosphofructokinase-1. After reassembly of both subunits in vitro a strong affinity of the reconstituted phosphofructokinase-1 to the dye-ligand was observed. The inability of the subunits to reconstitute under certain conditions seems to result from alterations of the intracellular environment following disruption. These changes give rise to induce an unproductive side reaction like self-aggregation of the subunits. Because reconstitution of phosphofructokinase-1 from S. cerevisiae behaves in a similar way to that of hemoglobin and luciferase, we would speculate a general mechanism for assembly of oligomeric proteins in vivo.
Topics: Blotting, Western; Chromatography, Affinity; Chromatography, High Pressure Liquid; Enzyme Reactivators; Enzyme-Linked Immunosorbent Assay; Genes, Fungal; Glucose; Molecular Weight; Phosphofructokinase-1; Protoplasts; Saccharomyces cerevisiae; Sequence Deletion
PubMed: 9559541
DOI: 10.1002/(SICI)1097-0061(19980315)14:4<323::AID-YEA223>3.0.CO;2-W -
Methods in Molecular Biology (Clifton,... 2014The evaluation of enzyme activities, especially their capacities, represents an important step towards the modelling of biochemical pathways in living organisms. The...
The evaluation of enzyme activities, especially their capacities, represents an important step towards the modelling of biochemical pathways in living organisms. The implementation of microplate technology enables the determination of up to >50 enzymes in relatively large numbers of samples and in various biological materials. Most of these enzymes are involved in central metabolism and several pathways are entirely covered. Direct or indirect assays can be used, as well as highly sensitive assays, depending on the abundance of the enzymes under study. To exemplify such methods, protocols for UDP-glucose pyrophosphorylase (E.C. 2.7.7.9) operating in real time and for pyrophosphate:fructose-6-phosphate 1-phosphotransferase (E.C. 2.7.1.90) are presented.
Topics: Diphosphates; Enzyme Assays; Kinetics; Phosphofructokinase-1; Plant Proteins; Plants; Reference Standards; Solutions; UTP-Glucose-1-Phosphate Uridylyltransferase
PubMed: 24222420
DOI: 10.1007/978-1-62703-688-7_15 -
Biochemistry Apr 1980
Topics: Adenosine Monophosphate; Adenosine Triphosphate; Animals; Enzyme Activation; Hydrogen-Ion Concentration; Kinetics; Liver; Magnesium; Phosphofructokinase-1; Rats; Temperature
PubMed: 6446316
DOI: 10.1021/bi00548a034 -
The Journal of Biological Chemistry Jun 2001Two phosphofructokinase genes have been described previously in Entamoeba histolytica. The product of the larger of the two genes codes for a 60-kDa protein that has...
Two phosphofructokinase genes have been described previously in Entamoeba histolytica. The product of the larger of the two genes codes for a 60-kDa protein that has been described previously as a pyrophosphate (PP(i))-dependent enzyme, and the product of the second, coding for a 48-kDa protein, has been previously reported to be a PP(i)-dependent enzyme with extremely low specific activity. Here it is found that the 48-kDa protein is not a PP(i)-dependent enzyme but a highly active ATP-requiring enzyme (k(cat) = 250 s(-)1) that binds the cosubstrate fructose 6-phosphate (Fru-6-P) with relatively low affinity. This enzyme exists in concentration- and ATP-dependent tetrameric active and dimeric inactive states. Activation is achieved in the presence of nucleoside triphosphates, ADP, and PP(i), but not by AMP, P(i), or the second substrate Fru-6-P. Activation by ATP is facilitated by conditions of molecular crowding. Divalent cations are not required, and no phosphoryl transfer occurs during activation. Kinetics of the activated enzyme show cooperativity with Fru-6-P (Fru-6-P(0.5) = 3.8 mm) and inhibition by high ATP and phosphoenolpyruvate. The enzyme is active without prior activation in extracts of E. histolytica. The level of mRNA, the amount of enzyme protein, and the enzyme activity of the 48-kDa enzyme are about one-tenth that of the 60-kDa enzyme in extracts of E. histolytica trophozoites.
Topics: Adenosine Triphosphate; Animals; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Entamoeba histolytica; Enzyme Activation; Kinetics; Phosphofructokinase-1; Recombinant Proteins
PubMed: 11262402
DOI: 10.1074/jbc.M011584200