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At least 19 recordsLinked to original sources

PubMed1999

Phosphofructokinase activities within the order spirochaetales and the characterisation of the pyrophosphate-dependent phosphofructokinase from spirochaeta thermophila

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, paragraph signT. 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 paragraph signL. 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.

Journal Article↗

PubMed1977

Allosteric and non-allosteric phosphofructokinases from Lactobacilli. Purification and properties of phosphofructokinases from L. plantarum and L. acidophilus.

Phosphofructokinase (ATP : D-fructose-6-phosphate 1 phosphotransferase, EC 2.7.1.11) from two different lactobacilli, Lactobacillus plantarum and Lactobacillus acidophilus were isolated and purified. Both enzymes have a molecular weight of 154 000 and consist of four subunits of identical size. Antisera from sheep immunized against the purified phosphofructokinase from L. plantarum showed immunologic cross reaction with the enzyme from L. acidophilus. In spite of the close molecular relationship indicated by the immunologic cross reaction, the kinetic behaviour of the two enzymes was strikingly different. Phosphofructokinase from L. plantarum showed pure Michaelis-Menten behaviour. Phosphofructokinase from L. acidophilus, however, showed sigmoidal substrate saturation curves for fructose 6-phosphate in the presence of slightly alkaline pH and high ATP concentrations; it was activated by fructose 1,6-biphosphate and inhibited by ADP. The results indicate that even enzymes which are structurally very similar may differ greatly with respect to their kinetic and regulatory properties and suggest that allosteric and non-allosteric phosphofructokinases have the same origin in evolution.

Adenosine Diphosphate↗

PubMed1980

Purification of F4 phosphofructokinase from human platelets and comparison with the other phosphofructokinase forms.

The phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) tetramers F4, F3L and F2L2 have been separated from human platelets, and purified to homogeneity by affinity chromatography on Dextran Blue-Sepharose 4B. The F subunits have a molecular weight of 85 000, identical to that of the M subunits. By contrast with L-type phosphofructokinase, the F-type enzyme seems to exist predominantly in a tetrameric form and not to aggregate to high molecular weight polymers. Specific activity of pure F4 phosphofructokinase is about 140 IU/mg of protein. Immunologically, it is easy to distinguish all the basic phosphofructokinase forms (i.e. M, L and F types); nevertheless a slight immunological cross-reactivity seems to exist between all these forms.

Blood Platelets↗

PubMed1988

Modulation of phosphofructokinase action by macromolecular interactions. Quantitative analysis of the phosphofructokinase-aldolase-calmodulin system.

The simultaneous effect of calmodulin and aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) on the concentration-dependent behaviour of muscle phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been analysed by means of a covalently attached fluorescent probe, gel penetration experiments, and using a kinetic approach. We found that calmodulin-induced inactivation of phosphofructokinase is suspended by addition of an equimolar amount of aldolase. This effect was attributed to an apparent competition of calmodulin and aldolase for the dimeric forms of kinase. Moreover, the direct binding of aldolase to calmodulin has also been demonstrated, which resulted in a significant decrease in the kcat value of the enzyme. The quantitative analysis of these interactions in the system phosphofructokinase-calmodulin-aldolase is presented. A possible molecular model for the modulation of phosphofructokinase action by macromolecular interactions is envisaged.

Animals↗

PubMed1986

Phosphofructokinase from the epithelial cells of rat small intestine. Comparison of regulatory properties with those of skeletal muscle, liver and brain phosphofructokinase.

The regulatory properties of phosphofructokinase from rat mucosa, liver, brain and muscle were investigated. Mucosal phosphofructokinase displayed cooperativity with respect to fructose 6-phosphate at pH 7.0 and so did the muscle, brain and liver isoenzymes. All these four isoenzymes were inhibited by ATP, the mucosal isoenzyme being the least inhibited. They were also inhibited by citrate and creatine phosphate. AMP, ADP, glucose 1,6-diphosphate, fructose 2,6-bisphosphate and inorganic phosphate were all strong activators for the mucosal, brain, liver and muscle phosphofructokinase, but the mucosal isoenzyme was found to be more activated than the others, accounting for the higher rates of glycolysis observed in mucosa. The results suggest that mucosal phosphofructokinase is unique and different from all the other isoenzymes.

Animals↗

PubMed1975

Interaction of phosphofructokinase with antibodies. Kinetic properties of phosphofructokinase in complexes with antibodies.

The allosteric properties of phosphofructokinase (EC 2.7.1.11) from rabbit muscle are influenced by enzyme concentration, most probably due to changes in the association state of the enzyme. In this study, the behaviour of dispersed pre-cipitates of phosphofructolinase as produced by treatment with antibodies has been investigated. The enzyme is not capable of rapid dissociation in the precipitated state as is confirmed by the lack of inactivation upon dilution and by the absence of shifts in substrate saturation curves as measured in the presence of different concentrations of the enzyme. The Hill coefficient of phosphofructokinase is decreased from 1.96 to 1.04 by antibody treatment. The V at neutral pH is increased 3-fold while the K0.5 for fructose 6-phosphate is reduced significantly. On the other hand, antibody-treated phosphofructokinase retains its sensitivity to allosteric activation by glucose 1,6-bisphosphate in the rpesence of high ATP concentrations.

Allosteric Regulation↗

PubMed1981

Phosphofructokinases from Lactobacteriaceae. II. Purification and properties of phosphofructokinase from Streptococcus thermophilus.

Phosphofructokinase (ATP : D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from Streptococcus thermophilus has been purified. It is a tetramer composed of identical subunits of molecular weight 36 000 and exhibits Michaelis-Menten kinetics. Compared to the phosphofructokinases from taxonomically related bacteria, the enzyme from S. thermophilus is more stable at high temperatures. In addition, it has been demonstrated that the phosphofructokinases from lactobacteria and also from Bacillus stearothermophilus show immunologic cross-reaction. In spite of the significantly different kinetic properties and the different thermostability of these enzymes, this finding indicates great structural resemblance.

Geobacillus stearothermophilus↗

PubMed1986

Aspects on the phosphorylation of muscle phosphofructokinase by protein kinase C--inhibition by phosphofructokinase stabilisers.

Our report presents data on the phosphorylation of muscle phosphofructokinase by Ca2+-activated, phospholipid-dependent protein kinase. We have found a stoichiometrical phosphorylation (about 1.5 mol per mol subunit), and a low apparent Km (about 0.7 microM). These data speak in favor of a physiological role for the reaction, as does the fact that phosphofructokinase from a new species (rat) was successfully phosphorylated. On the other hand we present the hitherto unpublished circumstance that the phosphorylation is inhibited by conditions that stabilise the activity of phosphofructokinase. This fact makes us question the true significance of this reaction.

Adenosine Monophosphate↗

PubMed1975

Erythrocyte phosphofructokinase deficiency associated with an unstable variant of muscle phosphofructokinase.

A case of chronic non-spherocytic hemolytic anemia due to partial erythrocyte phosphofructokinase deficiency (61% of normal) is reported. Immunological studies in hemolystates, using anti-muscle and anti-leukocyte phosphofructokinase antisera, seemed to indicate that an isozyme of the muscle type was deficient in the patient. This hypothesis was confirmed by the studies of muscle phosphofructokinase; this enzyme was an unstable and fast variant. There was no deficiency in muscle because of the active synthesis of proteins by this tissue, but the deficiency could be detected in erythrocytes, old cells which are no longer able to synthesize proteins.

Adolescent↗

PubMed1996

Activation by phosphorylation of phosphofructokinase from the annelid Lumbricus terrestris and comparison of phosphorylated sites in invertebrate phosphofructokinases.

Purified phosphofructokinase from the earthworm Lumbricus terrestris was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase from the same organism to an extent of approx. 0.5 mol/mol of subunit. Activation of the enzyme occurred in parallel to the incorporation of covalently bound phosphate and was reversed by the action of the catalytic subunit of protein phosphatase 2A. Phosphorylation decreased the co-operativity of fructose 6-phosphate saturation in the presence of inhibitory concentrations of ATP, and increased the apparent Vmax obtained with saturating concentrations of the activators 5'-AMP and fructose 2,6-bisphosphate. The phosphorylated sites of phosphofructokinase from L. terrestris and from two molluscs (Helix pomatia and Mytilus edulis) were sequenced and shown to exhibit distinct similarity to sequences located near to the N-terminus of nematode phosphofructokinases [Klein, Olson, Favreau, Wintertowed, Hatzenbuhler, Shea, Nulf and Geary (1991) Mol. Biochem. Parasitol. 48, 17-26.

Allosteric Regulation↗

PubMed1990

Genetic defect in muscle phosphofructokinase deficiency. Abnormal splicing of the muscle phosphofructokinase gene due to a point mutation at the 5'-splice site.

The genetic defect in muscle phosphofructokinase deficiency (type VII glycogenosis, Tarui disease) was investigated. Six cDNAs for muscle phosphofructokinase, including a full-length clone, were isolated from a non-amplified library of muscle from a patient. By sequence analysis of these clones, a 75-base in-frame deletion was identified. The rest of the sequence was identical to that of the normal cDNA, except for a silent base transition at position 516 (ACT (Thr) to ACC (Thr]. The deletion was located in the 3'-terminal region of exon 13 (numbered with reference to the rabbit muscle phosphofructokinase gene (Lee, C.-P., Kao, M.-C., French, B.A., Putney, S.D., and Chang, S.H. (1987) J. Biol. Chem. 262, 4195-4199]. Genomic DNA of the patient was amplified by polymerase chain reaction. Sequence analysis of the amplified DNA revealed a point mutation from G to T at the 5'-end of intron 13. This mutation changed the normal 5'-splice site of CAG:GTATGG to CAG:TTATGG. A cryptic splice site of ACT:GTGAGG located 75 bases upstream from the normal splice site was recognized and spliced in the patient.

Base Sequence↗

PubMed1984

Kinetic properties of erythrocyte phosphofructokinase in patients with type VII glycogenosis from two families--close similarity to liver type phosphofructokinase.

The kinetic properties of phosphofructokinases (PFKs) from normal human liver, muscle and erythrocytes, and from erythrocytes of two unrelated patients with type VII glycogenosis (muscle PFK deficiency, McKusick 23280) were analysed in this study. Sensitivity to inhibition by ATP and to inhibition by 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate and citrate were quite different for muscle and liver PFKs. The kinetic characteristics of normal erythrocyte PFK were intermediate between those of muscle and liver PFKs. The kinetic constants of erythrocyte PFK of a patient in one family were indistinguishable from those in the other family. In addition, kinetic behaviour of residual PFK activity in erythrocytes from patients in the two families were quite similar to those of normal liver PFK. These results of kinetic analyses provide convincing evidence for the concept that normal erythrocyte PFK consists of muscle and liver type subunits. Residual erythrocyte PFK activity in type VII glycogenosis is thus concluded to reflect the activity of liver type PFK existing in patient's erythrocytes.

Adolescent↗

PubMed2001

Sequence, expression, and characterization of the first archaeal ATP-dependent 6-phosphofructokinase, a non-allosteric enzyme related to the phosphofructokinase-B sugar kinase family, from the hyperthermophilic crenarchaeote Aeropyrum pernix.

The gene (ORF APF0012) encoding the ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic archaeon Aeropyrum pernix was identified, cloned, and functionally expressed in Escherichia coli. The deduced amino acid sequence showed similarity (25-40%) to members of PFK-B sugar kinases. The purified recombinant enzyme is a heterotetramer of 115 kDa, composed of 34-kDa subunits. Rate dependence (at 85 degrees C) on both fructose 6-phosphate (F-6-P) and ATP followed Michaelis-Menten kinetics with apparent K(m) values of 0.25 mM and 0.68 mM, respectively; apparent V(max) values were about 5 U/mg. The enzyme was specific for ATP as phosphoryl donor, but showed a broader spectrum of phosphoryl acceptors: in addition to F-6-P, glucose 6-phosphate, adenosine, fructose, ribose 5-phosphate, and ribose were accepted. Enzyme activity required divalent cations; Mg(2+), which was most effective, could partially be replaced by Co(2+), Ni(2+), or Mn(2+). The enzyme had a temperature optimum of 90 degrees C and showed a significant thermostability up to 100 degrees C. ATP-PFK activity was not allosterically regulated by classical effectors of ATP-PFKs of eukarya and bacteria, such as ADP and phosphoenolpyruvate. In accordance, this archaeal ATP-PFK did not contain the typical conserved binding sites for these effectors. This is the first report of a sequence of an archaeal ATP-PFK related to the PFK-B sugar kinase family.

Adenosine Triphosphate↗

PubMed1996

Characterization of expression of phosphofructokinase isoforms in isolated rat pancreatic islets and purified beta cells and cloning and expression of the rat phosphofructokinase-A isoform.

Phosphofructokinase (PFK) plays a key role in regulating glycolytic flux, and the mammalian enzyme is a tetramer. Three monomeric isoforms are encoded by separate genes, are differentially expressed in specific tissues, and are designated by tissues in which they are most abundant (A, muscle; B, liver; and C, brain). Glucose-induced insulin secretion from pancreatic islets requires glucose transport into islet beta-cells and glycolytic metabolism. Little is known about islet PFK isozymes, but the possibility that PFK-A is expressed in beta-cells is of interest because that isoform is thought to govern glycolytic oscillations and to interact with a metabolically activated beta-cell phospholipase A2 enzyme. Using as probe a PCR product generated from rat islet RNA with primers designed from the human PFK-A sequence, we have cloned a full-length PFK-A cDNA from a rat islet cDNA library. The rat PFK-A deduced amino-acid sequence is 96% identical to that of human PFK-A, and all residues thought to participate in substrate or allosteric effector binding are conserved between the two sequences. The rat PFK-A amino-acid sequence is 69% and 68% identical to those for rat PFK-B and rat PFK-C, respectively, and differences in residues involved in binding of allosteric effectors were observed among the three isoforms. Rat PFK-A expressed as a glutathione-S-transferase fusion protein was recognized by antibodies raised against a peptide in the PFK-A sequence. Expression of PFK isoform mRNA species was examined by RT-PCR in rat islets, in purified populations of beta-cells prepared by fluorescence-activated cell sorting (FACS), and in RIN-m5F insulinoma cells, all of which expressed mRNA species for PFK-A, -B, and -C isoforms. PFK-A mRNA was expressed at much lower levels in an islet alpha-cell-enriched population. Interleukin-1 impairs islet glucose metabolism and insulin secretion and was found to induce a specific decline in islet expression of PFK-A mRNA. These findings establish the sequence of rat PFK-A, demonstrate that it is expressed in FACS-purified islet beta-cells, and suggest that its expression is regulated by a cytokine which influences insulin secretion.

Amino Acid Sequence↗

PubMed2001

Creation of an allosteric phosphofructokinase starting with a nonallosteric enzyme. The case of dictyostelium discoideum phosphofructokinase.

An allosteric phosphofructokinase (PFK) was created by sequence manipulation of the nonallosteric enzyme from the slime mold Dictyostelium discoideum (DdPFK). Most amino acid residues proposed as important for catalytic and allosteric sites are conserved in DdPFK except for a few of them, and their reversion did not modify its kinetic behavior. However, deletions at the unique C-terminal extension of this PFK produced a markedly allosteric enzyme. Thus, a mutant lacking the last 26 C-terminal residues exhibited hysteresis in the time course, intense cooperativity (n(H) = 3.8), and a 200-fold decrease in the apparent affinity for fructose 6-phosphate (S(0.5) = 4500 microm), strong activation by fructose 2,6-bisphosphate (K(act) = 0.1 microm) and fructose 1,6-bisphosphate (K(act) = 40 microm), dependence on enzyme concentration, proton inhibition, and subunit association-dissociation in response to fructose 6-phosphate versus the nonhysteretic and hyperbolic wild-type enzyme (n(H) = 1.0; K(m) = 22 microm) that remained as a stable tetramer. Systematic deletions and point mutations at the C-tail region of DdPFK identified the last C-terminal residue, Leu(834), as critical to produce a nonallosteric enzyme. All allosteric mutants were practically insensitive to MgATP inhibition, suggesting that this effect does not involve the same allosteric transition as that responsible for fructose 6-phosphate cooperativity and fructose bisphosphate activation.

Adenosine Triphosphate↗

PubMed1983

Muscle phosphofructokinase deficiency. Biochemical and immunological studies of phosphofructokinase isozymes in muscle culture.

Muscle cultures from three unrelated patients with muscle phosphofructokinase (PFK; EC 2.7.1.11) deficiency (Glycogenosis type VII; Tarui disease) had normal PFK activity and normal morphology. Chromatographic and immunological studies showed that normal muscle cultures express all three PFK subunits, M (muscle-type), L (liver-type), and P (platelet-type) and contain multiple homotetrameric and heterotetrameric isozymes. Muscle cultures from patients lack catalytically active M subunit-containing isozymes, but this is compensated for by the presence of P- and L-containing isozymes. Despite the lack of muscle-type PFK activity, presence of immunoreactive M subunit was demonstrable by indirect immunofluorescence, suggesting a mutation of the structural gene coding for the M-subunit of PFK.

Adult↗

PubMed1977

In vivo lability of red cell phosphofructokinase in term infants: the possible molecular basis of the relative phosphofructokinase deficiency in neonatal red cells.

Cord blood erythrocytes from nine term infants were separated by density gradient centrifugation into cohorts of intact cells of progressively increasing density and compared with red cells treated in a similar manner from four healthy adults. Pyruvate kinase (PK), an age-dependent enzyme, progressively decreased in activity from the lightest to the heaviest fractions, in both neonatal and adult red cells, indicating that red cells from newborn infants exhibit the same relationship between red cell age and density that had previously been demonstrated in red cells from adults. The rate of decline of red cell PK activity was essentially the same in neonates and adults, whereas phosphofructokinase (PFK) activity in cord erythrocytes decreased at a significantly faster rate when compared to adults. These data suggest that PFK has an accelerated rate of in vivo decay in neonatal red cells and is an unstable enzyme in the newborn.

Adult↗

PubMed1995

Functional expression of human mutant phosphofructokinase in yeast: genetic defects in French Canadian and Swiss patients with phosphofructokinase deficiency.

Human phosphofructokinase (PFK) is a tetrameric enzyme, encoded by muscle, liver, and platelet genes. Deficiency of muscle PFK (PFK-M), glycogenosis type VII (Tarui disease), is an autosomal recessive disorder characterized by an exertional myopathy and hemolytic syndrome. Several disease-causing mutations have been identified in the PFK-M gene in Japanese, Ashkenazi Jewish, and Italian patients. We describe the genetic defects in French Canadian and Swiss patients with the disease, and we use a genetically well-defined yeast system devoid of endogenous PFK for structure-function studies of the mutant PFKs. A G-to-A transition at codon 209-in exon 8 of the PFK-M gene, changing an encoded Gly to Asp, is responsible for the disease in a homozygous French Canadian patient. Gly-209-mutated protein is completely inactive in the yeast system. The Swiss patient is a genetic compound, carrying a G-to-A transition at codon 100 in exon 6 (Arg to Gln) and a G-to-A transition at codon 696 in exon 22 (Arg to His). The mutants expressed in yeast generate functional enzyme with modest changes in thermal stability. The advantages and limitations of the yeast system for expression of human mutant PFKs are discussed.

Adult↗