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PubMed1986

Zn2+-dependent reversible inactivation of rat liver phosphofructokinase-1. Purification of the inactivating protein and characterization of the inactivation reaction.

A protein has been purified from rat liver (about 5 mg from 100 g) which inactivates rat liver phosphofructokinase-1. According to dodecyl sulfate gel electrophoresis the protein consists of a single peptide chain with a Mr of 19,000. The inactivation of phosphofructokinase-1 by this protein results from a dissociation of phosphofructokinase-1 into its inactive protomers (Mr = 82,000). The inactivation is dependent on zinc ions in micromolar concentration (about 1-2 microM), but is inhibited by higher concentrations (greater than 50 microM). Fructose 1,6-bisphosphate as well as fructose 2,6-bisphosphate inhibit the inactivation reaction. In addition, both compounds as well as ATP can reverse the dissociation of phosphofructokinase-1. The phosphofructokinase-1 inactivating protein has no phosphatase activity with [32P]phosphofructokinase or low molecular weight phospho-compounds and does not possess any detectable proteolytic activity. It has the same affinity for the phospho- and the dephosphoform of phosphofructokinase-1, but preincubation of phosphofructokinase-1 with this inactivating protein reduces the maximum amount of phosphate incorporated into phosphofructokinase-1 and accelerates the velocity of the dephosphorylation reaction. A direct Zn2+-dependent binding of phosphofructokinase-1 to the inactivating protein has been demonstrated in experiments with matrix-bound phosphofructokinase-1 inactivating protein.

Animals↗
PubMed1993

Limited proteolysis of yeast phosphofructokinase. Sequence locations of cleavage sites created by the actions of different proteinases.

Purified phosphofructokinase 1 from baker's yeast (Saccharomyces cerevisiae) was subjected to proteolysis by thermolysin, endoproteinase lys-C, trypsin and chymotrypsin under defined solvent conditions. In the absence of substrates and allosteric effectors, the catalytic activity of phosphofructokinase rapidly disappeared in the presence of each proteolytic enzyme. The presence of a saturating concentration of ATP protected phosphofructokinase activity from proteolytic inactivation while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate provided transient activation during proteolysis. Changes in the quaternary structure of phosphofructokinase resulting from proteolysis were estimated by high performance size exclusion chromatography while changes in the primary sequence of the individual alpha and beta polypeptide chains were estimated by polyacrylamide-gel electrophoresis in sodium dodecylsulfate. The site(s) of proteolytic cleavage were identified by N-terminal sequence analysis of resolved electrophoretic components. The presence of ATP protects phosphofructokinase from thermolysin proteolysis, while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate restricts proteolysis to one site in each polypeptide chain involving the peptide bonds preceding Leu199 in the alpha chain and Leu192 in the beta chain. The truncated phosphofructokinase retains its octameric structure. The presence of ATP largely restricts endoproteinase lys-C proteolysis to a single site in the alpha chain involving the peptide bond preceding Val914. This cleavage results in the dissociation of the octameric form of phosphofructokinase into two tetramers. The presence of ATP restricts both trypsin and chymotrypsin proteolysis to the N-terminal and C-terminal regions described above, resulting in the preferential stabilization of the tetrameric form of phosphofructokinase. It would appear that the first 200 and last 80 residues which are unique to the sequence of the yeast phosphofructokinase are not directly involved in catalysis or its allosteric regulation. However, the last 80 residues of the alpha polypeptide chain do appear to stabilize an octameric structure which is unique to yeast phosphofructokinase.

Adenosine Monophosphate↗
PubMed1988

Identification of two different phosphofructokinase-phosphorylating protein kinases from Ascaris suum muscle.

Two different phosphofructokinase-phosphorylating protein kinases were separated from extracts of Ascaris suum muscle by chromatography on DEAE-Fractogel. They were tentatively designated phosphofructokinase kinase I and phosphofructokinase kinase II. Phosphofructokinase kinase I eluted from the chromatography column at an ionic strength of 0.07 and contained about 25% of the phosphofructokinase-phosphorylating activity assayed in crude extracts. The protein kinase activity was not stimulated by the addition of either cAMP or cGMP. It was inhibited by the heat-stable protein kinase inhibitory protein from rabbit muscle (Walsh inhibitor), by the regulatory subunit of cAMP-dependent protein kinase from beef heart, and by the cAMP-binding protein from Ascaris muscle. These properties suggest that phosphofructokinase kinase I is homologous to the catalytic subunit of cAMP-dependent protein kinases from mammals. This assumption is supported by the estimation of the Mr of 40,000 for the purified phosphofructokinase kinase I under denaturing conditions and by the fact that the presence of cAMP eliminated the inhibition by the cAMP binding proteins. The isoelectric point of the enzyme was 8.7. Phosphofructokinase kinase II was eluted from the DEAE-Fractogel column at an ionic strength of 0.16 and contained approximately 75% of the phosphofructokinase kinase activity measured in the extracts. The molecular and kinetic properties were significantly different from those of phosphofructokinase kinase I. The enzyme was not inhibited by the heat-stable inhibitor protein nor by cAMP-binding proteins. The Mr of the native enzyme was estimated as 220,000 by molecular sieve chromatography. The isoelectric point of the enzyme was pH 5.45.

Animals↗
PubMed1989

Phosphofructokinase in alveolar type II cells isolated from fetal and adult rat lung.

The glycolytic enzyme 6-phosphofructokinase (EC 2.7.1.11) was studied in adult and fetal type II pneumocytes which had been isolated from rat lung at different days of development. In addition, the activities of the enzymes hexokinase (EC 2.7.1.1), enolase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were assayed. The specific activities of the latter enzymes decrease during perinatal development and reach about adult values shortly after birth. In contrast, 6-phosphofructokinase activity increases slightly until 2 days before birth, and drops sharply afterwards. The 6-phosphofructokinase subunit composition was determined in fetal and adult type II cells. The ratio of the three subunits of 6-phosphofructokinase in type II cells isolated on fetal days 19 and 21 (term is at day 22) and in adult type II cells was identical: the three subunits were present in a ratio of 68: 14: 18 for types L, M and C, respectively. In addition, we investigated some regulatory properties of 6-phosphofructokinase from alveolar type II cells. 6-Phosphofructokinase from alveolar type II cells is strongly inhibited by increasing MgATP concentrations. This inhibition is reflected by an increase in the S0.5 for fructose 6-phosphate. Fructose 2,6-bisphosphate stimulates alveolar type II 6-phosphofructokinase. Half-maximal stimulation occurs at 1.6 and 2.0 microM fructose 2,6-bisphosphate for fetal and adult type II cells, respectively. The level of the most potent positive effector of 6-phosphofructokinase, fructose 2,6-bisphosphate, was also determined. The level of the hexose bisphosphate decreases during prenatal development; however, the level in the adult type II cells is considerably lower. The concentration of fructose 2,6-bisphosphate appears to be sufficient to fully activate 6-phosphofructokinase both in fetal and adult type II cells.

Adenosine Triphosphate↗
PubMed1968

Metabolic control mechanisms in mammalian systems. Hormonal regulation of phosphofructokinase in the rat prostate and seminal vesicles.

1. The hormonal regulation of phosphofructokinase was investigated in the accessory reproductive organs of the orchidectomized rat. 2. Phosphofructokinase activities declined to 51% and 47% in the prostate and 9% and 6% of the normal values in seminal vesicles 4 and 8 weeks after castration respectively. Administration of testosterone (100mug./100g. body wt.) for 3 days reversed substantially the effects of orchidectomy, and phosphofructokinase activity increased to 173% in the prostate and 536% in seminal vesicles as compared with the values of castrated controls. 3. Time-course studies demonstrated that after a single injection of testosterone (5mg./100g. body wt.) phosphofructokinase activity was maximally elevated to 236% in the prostate and 342% in seminal vesicles at 24hr. 4. Dose-response studies revealed that 2.5mg. of testosterone propionate/100g. body wt. was the minimal amount necessary to induce significant increases in enzyme activity in both accessory sex organs; maximal increases were obtained with a dose of 5mg./100g. body wt. 5. The observed enzyme increases induced by testosterone were inhibited by the simultaneous administration of oestradiol-17beta, and phosphofructokinase activity in this group of rats remained at 97% in the prostate and 137% of the control values in seminal vesicles. Oestradiol-17beta by itself failed to produce any significant effect on enzyme activity in either of these secondary sexual tissues. 6. The nature of the testosterone-induced increases in phosphofructokinase activity was studied by using a variety of inhibitors of RNA and protein synthesis. Cycloheximide, 5-fluorouracil and ethionine largely blocked the androgen-stimulated rise in enzyme activity observed 24hr. after steroid injection. The inhibitory effect of ethionine was completely reversed by the simultaneous administration of methionine. 7. Actinomycin, which is known to inhibit the synthesis of messenger RNA as well as the synthesis of other cellular RNA fractions, when given simultaneously with the hormone, also inhibited the testosterone-induced increases in prostatic and seminal-vesicular phosphofructokinase. However, when the antibiotic was given 6 or 12hr. after injection of the steroid, practically no inhibition of phosphofructokinase induction was obtained. This indicates that, once the enzyme-forming machinery is turned on and allowed to operate for a few hours, actinomycin is incapable of reversing the hormone-induced enzyme responses. 8. The results presented suggest that new RNA and protein synthesis may be involved in the observed androgen-induced increases in phosphofructokinase activity in the prostate and seminal vesicles of the orchidectomized rat.

Animals↗
PubMed1975

The effects of ammonium, inorganic phosphate and potassium ions on the activity of phosphofructokinases from muscle and nervous tissues of vertebrates and invertebrates.

1. The effect of NH4+, Pi and K+ on phosphofructokinase from muscle and nervous tissues of a large number of animals was investigated. The activation of the enzyme from lobster abdominal muscle by NH4+ was increased synergistically by the presence of Pi or SO4(2-). In the absence of K+, NH4+ plus Pi markedly activated phosphofructokinase from all tissues studied. In the presence of 100 mM-K+, NH4+ plus Pi activated phosphofructokinase from nervous tissue and muscle of invertebrates and the enzyme from brain of vertebrates, but there was no effect of NH4+ plus Pi on the enzyme from the muscles of vertebrates. Nonetheless, NH4+ plus Pi increased the activity of vertebrate muscle phosphofructokinase in the presence of 50 mM-K+ at inhibitory concentrations of ATP, i.e. these ions de-inhibited the enzyme. In the absence of NH4+ plus Pi, K+ activated phosphofructokinase from vertebrate tissues at non-inhibitory ATP concentrations, but the effect was less marked with the enzyme from invertebrate tissues. Indeed, high concentrations of K+ (greater than 50 mM) caused inhibition of invertebrate tissue phosphofructokinase. Of the other alkali-metal ions tested, only Rb+ activated phosphofructokinase from lobster abdominal muscle and rat heart muscle. 2. The properties of lobster abdominal-muscle phosphofructokinase were studied in detail. This muscle was chosen as representative of invertebrate muscle because large quantities of tissue could be obtained from one animal and the enzyme was considerably more stable in tissue extracts than in extracts of insect flight muscle. In general, the properties of the enzyme from this tissue were similar to those of the enzyme from many other tissues: ATP concentrations above an optimum value inhibited the enzyme and this inhibition was decreased by raising the fructose 6-phosphate or the AMP concentration. In particular, NH4+ plus Pi activated the enzyme at noninhibitory concentrations of ATP and they also relieved ATP inhibition (see above). 3. It is suggested that increases in the concentration of NH4+ and Pi, under conditions of increased ATP utilization in certain muscles and/or nervous tissue, may play a part in the stimulation of glycolysis through the effects on phosphofructokinase (the effect may be a direct activation and/or a relief of ATP inhibition). Changes in the concentration of NH4+ and Pi are consistent with this theory in nervous tissue and the anaerobic type of muscles. The role of AMP deaminase in production of NH4+ from AMP in these tissues is discussed in relation to the control of glycolysis.

AMP Deaminase↗
PubMed1983

Ca2+-mediated activation of phosphofructokinase in perfused rat heart.

Perfusion of the isolated rat heart with Ca2+ concentrations exceeding 3 mM activated phosphofructokinase and phosphorylase, and decreased the concentration of cyclic AMP. Half-maximal activation of phosphofructokinase occurred at 5 mM-CaCl2; significant activation of phosphorylase did not occur until the concentration of CaCl2 exceeded 12 mM. The time course for the activation of phosphofructokinase at 12 mM-CaCl2 indicated that maximal activation occurred within 2 min; when the perfusion-medium Ca2+ concentration was re-adjusted to 3 mM, the phosphofructokinase activity returned to pre-activation values within 30 s. The addition of Ca2+ to extracts of heart did not activate phosphofructokinase. The activation of phosphofructokinase by sub-maximal doses of adrenaline and Ca2+ were not additive. The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol and by 1 microM-isoprenaline was inhibited by high concentrations of K+ (22-56 mM). The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol, 12 mM-CaCl2 and by 1 microM-isoprenaline was blocked by the slow Ca2+-channel blocker nifedipine. These findings suggest that both the beta- and alpha-adrenergic mechanisms for the activation of rat heart phosphofructokinase involve an increase in the myoplasmic Ca2+ concentration. This increase may result from an inhibition of Ca2+ efflux or a stimulation of Ca2+ influx.

Animals↗
PubMed1987

The effect of experimental hypothyroidism on phosphofructokinase activity and fructose 2,6-bisphosphate concentrations in rat heart.

Experimental hypothyroidism was induced in rats by the administration of NaClO4. Hearts from normal and hypothyroid rats were homogenized, and the extracts were assayed for phosphofructokinase-1 and phosphofructokinase-2 activity and fructose 2,6-bisphosphate concentrations. Hypothyroidism was associated with a drastic loss of phosphofructokinase-1 activity. A hyperbolic relationship between plasma thyroxine concentrations and phosphofructokinase-1 activity was found. As treatment with NaClO4 progressed, the decrease in blood thyroxine was faster than the decrease in enzyme activity. After prolonged hypothyroidism (a decrease in thyroxine of more than 10-fold), a 4-fold decrease in phosphofructokinase-1 activity was observed. In this metabolic condition 2-fold decreases in phosphofructokinase-2 activity and in fructose 2,6-bisphosphate were observed. A similar decrease in phosphofructokinase-1 activity in a partially purified preparation was found. The addition of L-thyroxine in the diet had little effect on phosphofructokinase-1 activity. However, exposure of minced pieces of hearts of hypothyroid rats to tri-iodothyronine for 5 h resulted in a clear increase in phosphofructokinase-1 activity, which was partially prevented by the simultaneous addition of cycloheximide. These results could account for the decrease in carbohydrate metabolism in heart from hypothyroid rats.

Adenosine Monophosphate↗
PubMed1981

Regulatory properties of phosphofructokinase 2 from Escherichia coli.

Escherichia coli K12 contains two phosphofructokinases: phosphofructokinase 1, the most studied one, appears to behave as an allosteric enzyme, while phosphofructokinase 2 presents the features of a Michaelian enzyme. We show the present paper that, in fact, phosphofructokinase 2 also presents some regulatory properties in vitro: at high concentrations, ATP is an inhibitor of phosphofructokinase 2 and it provokes the tetramerization of the dimeric native enzyme. The binding of the two substrates to phosphofructokinase 2 is sequential and ordered as for phosphofructokinase 1, but in the former case fructose 6-phosphate is the first substrate to be bound and ADP the first product to be released. Each dimer of phosphofructokinase 2 binds two molecules of fructose 6-phosphate but only one molecule of the product fructose 1,6-phosphate. Although both phosphofructokinases of E. coli K12 present regulatory properties in vitro, the mechanism of regulation of the activity of the two enzymes is strikingly different. It can be asked whether or not these mechanisms operate in vivo.

Escherichia coli↗
PubMed1986

Purification and properties of spinach leaf phosphofructokinase 2/fructose 2,6-bisphosphatase.

Phosphofructokinase 2 was purified from spinach leaves by fractionation with poly(ethylene glycol) and by chromatography on blue Sepharose, anion exchanger Mono-Q and blue Trisacryl. A low-Km fructose-2,6-bisphosphatase copurified with phosphofructokinase 2 and its constitutive subunits could be easily identified by sodium dodecyl sulphate gel electrophoresis thanks to the formation of a [32P]phosphoenzyme intermediate upon short-time incubation in the presence of 1 microM fructose 2,6-[2-32P]bisphosphate. On anion-exchange chromatography, two peaks of phosphofructokinase 2/fructose-2,6-bisphosphatase were resolved. The first one, called L (light), represented about 10% of the phosphofructokinase 2 activity and was characterized by a phosphofructokinase 2/fructose-2,6-bisphosphatase activity ratio close to 1, by an Mr of 132,000 as measured by gel filtration, and by a series of subunits of Mr comprised between 44,000 and 70,000. The second and major peak of phosphofructokinase 2, called H (heavy), had a phosphofructokinase 2/fructose-2,6-bisphosphatase ratio close to 8, an Mr of 390,000 and was made of 90,000-Mr subunits. The H form of phosphofructokinase 2 had a lower Km for fructose 6-phosphate than the L form and a higher Ki for a series of physiological inhibitors. By contrast, the kinetics of fructose-2,6-bisphosphatase was the same for the two forms of the enzyme. Upon incubation in the presence of papain or of a crude spinach leaf extract, the purified H form gave rise to products made of subunits of Mr comprised between 70,000 and 44,000 but also of lower values which maintained their fructose-2,6-bisphosphatase activity. The H and L forms of phosphofructokinase 2/fructose-2,6-bisphosphatase were also detected in crude homogenates of castor bean endosperm and of Jerusalem artichoke tubers.

Animals↗
PubMed1985

Mode of interaction of phosphofructokinase with the erythrocyte membrane.

Phosphofructokinase is known to associate with the human erythrocyte membrane both in vitro and in vivo. Such association activates the enzyme in vitro by relieving the allosteric inhibition imposed by ATP (Karadsheh, N.S., and Uyeda, K. (1977) J. Biol. Chem. 252, 7418-7420). We now demonstrate that ADP, ATP, and NADH, all of which are known to bind to the enzyme's adenine nucleotide activation site, are particularly potent in eluting the enzyme from the membrane. In addition, both inside-out red cell membrane vesicles and a 23-kDa fragment containing the amino terminus of the membrane protein, band 3, cause a slow, partial, and reversible inactivation of phosphofructokinase. The dependence of the residual phosphofructokinase activity on phosphofructokinase concentration demonstrates that inactivation occurs through the dissociation of active tetramers to inactive dimers. Dimers of phosphofructokinase associate with the membrane more avidly than tetramers. The kinetics of phosphofructokinase inactivation are consistent with the dissociation of tetramers in solution followed by the binding of dimers to the membrane. There is no indication of an association equilibrium between tetramers and dimers of phosphofructokinase bound to the membrane. Taken together, these results suggest that the amino-terminal segment of band 3 binds to the adenine nucleotide activation site, which is thought to be located in a cleft between the dimeric subunits of phosphofructokinase. As a result, band 3 not only rapidly activates the phosphofructokinase tetramer but also slowly inactivates the enzyme by preferentially binding its dissociated subunits.

Acetylcholinesterase↗
PubMed1991

Hepatic phosphofructokinase-1 activity and fructose 2,6-bisphosphate levels in patients with abdominal sepsis.

1. In sepsis various processes of carbohydrate metabolism, such as hepatic gluconeogenesis and glycolysis, are altered. Phosphofructokinase-1, a key glycolytic enzyme, is controlled in the long term via regulation of synthesis and degradation of the protein itself, while in the short term it is regulated by allosteric effectors, such as fructose 2,6-bisphosphate (the most potent). In the present study hepatic phosphofructokinase-1 activity as well as phosphofructokinase-2 activity and the concentration of fructose 2,6-bisphosphate were assayed to determine if they might contribute to the derangement of carbohydrate metabolism seen commonly in sepsis. 2. The levels of glycogen and fructose 2,6-bisphosphate and the activity of phosphofructokinase-1 and phosphofructokinase-2 were determined in hepatic biopsies obtained at laparotomy from six patients with and seven patients without abdominal septic foci. 3. A significant increase in plasma lactate concentration was observed in the septic patients, whereas no significant differences in tissue glycogen content or plasma glucose concentration were seen between the groups. 4. No significant change in plasma insulin concentration was observed. However, levels of the counter-regulatory hormones (glucagon, cortisol and adrenaline) were elevated in the septic patients. 5. A 60% decrease in hepatic phosphofructokinase-1 activity was seen in the septic patients. However, no significant changes in hepatic phosphofructokinase-2 activity and fructose 2,6-bisphosphate content were observed in the septic patients. 6. The present results demonstrate that the decrease in hepatic phosphofructokinase-1 activity occurring in sepsis does not appear to reflect alterations in the concentration of fructose 2,6-bisphosphate.

Abdomen↗
PubMed1997

Overexpression of phosphofructokinase and pyruvate kinase in citric acid-producing Aspergillus niger.

Phosphofructokinase and pyruvate kinase were overexpressed in the filamentous fungus Aspergillus niger. Moderate overexpression of these glycolytic enzymes in A. niger N400 (3-5-fold the wild-type level), either individually or simultaneously, did not increase citric acid production by the fungus significantly. Thus, phosphofructokinase and pyruvate kinase do not seem to contribute in a major way to flux control of the metabolism involved in the conversion of glucose to citric acid. Overexpression of phosphofructokinase and pyruvate kinase did not influence the activities of other enzymes in the pathway, nor did it change intermediary metabolite levels. However, in strains overexpressing phosphofructokinase, the level of fructose 2,6-bisphosphate, a positive allosteric effector of phosphofructokinase, was reduced almost 2-fold compared to the wild-type strain. Measurements with purified phosphofructokinase, using substrate, product and effector concentrations found intracellularly, showed that such a reduction in the fructose-2,6-bisphosphate level could decrease the specific activity of phosphofructokinase in the cell significantly. Thus, the fungus seems to adapt to overexpression of phosphofructokinase by decreasing the specific activity of the enzyme through a reduction in the level of fructose 2,6-bisphosphate.

Aspergillus niger↗
PubMed1983

The effect of starvation on the control of phosphofructokinase activity in the epithelial cells of the rat small intestine.

1. The effect of depriving rats of food for 48 h on the specific activity of phosphofructokinase in the epithelial cells of the small intestine and on the regulatory properties of the enzyme displayed in crude (particle-free) mucosal extracts was studied. 2. The specific activity of phosphofructokinase, measured under optimal conditions at pH8, in the mucosa of fed rats showed a negative aboral gradient along the intestine, decreasing from 15.2 +/- 1.2 units (mumol/min)/g wet wt. in the proximal jejunum to 4.6 +/- 1.2 units/g wet wt. in the terminal ileum. 3. After starvation, the gradient was diminished, but not abolished; the diminution in gradient was due almost exclusively to a decrease in the specific activity of phosphofructokinase in the proximal jejunum by about 30%, there being no change in the terminal ileum. 4. In fed rats, the susceptibility of phosphofructokinase to inhibition by ATP, when assayed in crude mucosal extracts under suboptimal conditions, was independent of length along the small intestine; the ratio of the activity observed at pH 7.0 in the presence of 0.5 mM-fructose 6-phosphate and 2.5 mM-ATP to the optimal activity at pH 8, v0.5/V, was 0.36 +/- 0.05 in the proximal jejunum and 0.42 +/- 0.07 in the terminal ileum. 5. After starvation, the susceptibility of phosphofructokinase to inhibition by ATP was increased and was again found to be independent of length along the small intestine: after starvation, v0.5/V was 0.19 +/- 0.04 and 0.20 +/- 0.07 for the proximal jejunum and the terminal ileum respectively. 6. Re-feeding of previously starved rats on a high-carbohydrate diet overnight for 16 h restored both the specific activities of phosphofructokinase and its susceptibility to inhibition by ATP to normal values for fed rats. 7. The data support the idea that the specific activities and the regulatory properties of phosphofructokinase in the epithelial cells of rat small intestine are mediated by distinct humoral factors. 8. The changes in glucose utilization rate of the jejunum when rats are starved can in principle be accounted for by a combination of changes in the specific activity and in the regulatory properties of mucosal phosphofructokinase.

Animals↗
PubMed1984

Insensitivity of cardiac phosphofructokinase to adrenergic activation in Zucker rats. A post-receptor defect.

The apparent insensitivity of phosphofructokinase to activation by adrenaline in hearts from genetically obese Zucker (fa/fa) rats [Patten, Filsell & Clark (1982) Metab. Clin. Exp. 31, 1137-1141] was examined in detail. Perfusion of hearts from obese (fa/fa) animals with medium containing 1nM-100 microM-adrenaline for 4 min did not significantly activate phosphofructokinase, but fully activated glycogen phosphorylase. Activation of phosphofructokinase occurred in hearts from lean (Fa/?) animals, with half-maximal activation at 10nM-adrenaline. Binding characteristics of cardiac alpha 1-adrenergic receptors were comparable between lean and obese animals. Properties of phosphofructokinase from obese and lean hearts were similar. High extracellular Ca2+ concentration, which activated phosphofructokinase in hearts from lean animals and phosphorylase in all hearts, failed to activate phosphofructokinase in hearts from obese rats. The data indicate that the failure of adrenaline to activate phosphofructokinase in hearts from obese (fa/fa) rats results from a post-receptor defect in the as-yet unidentified Ca2+-dependent phosphofructokinase-activating reactions.

Animals↗
PubMed1997

A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli.

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.

Carbon Dioxide↗
PubMed1988

Immunological and biochemical studies on the relationship between two actin-binding proteins, phosphofructokinase and gelsolin.

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.

Actins↗
PubMed1986

Purification and identification of intermediate catabolic products in the in vivo degradation of pig liver phosphofructokinase.

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified to homogeneity from pig livers. Polyclonal antibody against the enzyme was induced in a rabbit, and the IgG fraction was obtained by chromatography on a Protein A-Sepharose CL-4B column. The specific antibody was purified further by immunoaffinity chromatography on a phosphofructokinase-conjugated affinity column. Intermediate catabolic products of phosphofructokinase were extracted from fresh pig livers under conditions of inhibition of proteinases, concentrated by chromatography on an anti-phosphofructokinase IgG-conjugated affinity column, and purified by two-dimensional polyacrylamide gel electrophoresis. Their cross-reactivities to the purified phosphofructokinase were assessed by an immunoelectrotransfer blot method. The intact form of phosphofructokinase in pig liver was demonstrated as the major spot of 84 kDa on the blot. Polypeptides of 68, 64, 56, and 51 kDa showed apparent cross-reactivities to phosphofructokinase. The structural homology among them was confirmed by proteinase V8 digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possibility of artifacts in preparation was ruled out by an internal tracer method. Thus, it is concluded that the predominant isozyme of phosphofructokinase in pig liver (84 kDa) is in vivo degraded via intermediate catabolic products of 68, 64, 56, and 51 kDa.

Animals↗