Stereospecificity of the fructose 2,6-bisphosphate site of muscle 6-phosphofructo-1-kinase

We explored the stereospecificity of the fructose 2,6-bisphosphate site of rabbit muscle 6-phosphofructo-1-kinase by determination of the activation constants (Ka) of several structurally locked analogues of this potent metabolic regulator. Under the assay conditions used, the Ka of fructose 2,6-bisphosphate was 0.12 microM. The most effective synthetic analogues and their Ka's were 2,5-anhydro-D-mannitol 1,6-bisphosphate (2.9 microM), 1,4-butanediol bisphosphate (6.6 microM), hexitol 1,6-bisphosphate (40 microM), and 2,5-anhydro-D-glucitol 1,6-bisphosphate (47 microM). Ten other bisphosphate compounds were much less effective as activators of the enzyme. These findings indicate that, unlike its active site, this allosteric site of 6-phosphofructo-1-kinase does not require the furanose ring. Its basic requirement seems to be a compound with two phosphate groups approximately 9 A apart. Although the free hydroxy groups of the activator do not seem to be essential, their presence enhances appreciably the affinity of the ligand for this regulatory site.     

Purification and kinetic properties of 6-phosphofructo-1-kinase from gilthead sea bream muscle

The kinetic properties of 6-phosphofructo-1-kinase (PFK) from skeletal muscle (PFKM) of gilthead sea bream (Sparus aurata) were studied, after 10,900-fold purification to homogeneity. The native enzyme had an apparent molecular mass of 662 kDa and is composed of 81 kDa subunits, suggesting a homooctameric structure. At physiological pH, S. aurata PFKM exhibited sigmoidal kinetics for the substrates, fructose-6-phosphate (fru-6-P) and ATP. Fructose-2,6-bisphosphate (fru-2,6-P(2)) converted the saturation curves for fru-6-P to hyperbolic, activated PFKM synergistically with other positive effectors of the enzyme such as AMP and ADP, and counteracted ATP and citrate inhibition. The fish enzyme showed differences regarding other animal PFKs: it is active as a homooctamer, and fru-2,6-P(2) and pH affected affinity for ATP. By monitoring incorporation of (32)P from ATP, we show that fish PFKM is a substrate for the cAMP-dependent protein kinase. The mechanism involved in PFKM activation by phosphorylation contrasts with previous observations in other species: it increased V(max) and did not affect affinity for fru-6-P. Unlike the mammalian muscle enzyme, our findings support that phosphorylation of PFKM may exert a major role during starvation in fish muscle.     

Identification of two forms of 6-phosphofructo-2-kinase in yeast

Two forms of 6-phosphofructo-2-kinase have been identified in Saccharomyces cerevisiae by their different chromatographic behaviour on CM-Sephadex C-50. One of them was not adsorbed and represented approximately 30% of the eluted activity. The other one emerged at about 120 mM KCl. A molecular mass of 120 kDa was found for both of them. No differences in kinetic behaviour in susceptibility to activation by cAMP-dependent protein kinase were found between the two forms.     

Insulin-mediated regulation of heart atrial and ventricular 6-phosphofructo-1-kinase

Atrial 6-phosphofructo-1-kinase activity from the hearts of diabetic rats was decreased by 50%, but ventricular 6-phosphofructo-1-kinase activity was found not to be insulin-sensitive. This decrease in atrial 6-phosphofructo-1-kinase activity during diabetes was characterized by diminished levels of all three types of 6-phosphofructo-1-kinase subunits. As shown by immunological titration and column chromatography, the population of native 6-phosphofructo-1-kinase isozymes in the ventricles was not measurably affected during insulin deprivation. However, the atrial isozyme population in diabetic rat heart appeared to contain, on a relative basis, higher levels of the isozymic forms containing the L-type subunit. Measurement of the levels of this subunit indicated that in diabetic atria it was less affected than the other subunits. In the ventricles, insulin deficiency did not promote significant losses of fructose-2,6-P2; but, in diabetic rats, the atrial levels of this activator were decreased by 80% and subsequently restored by insulin treatment. These data suggest that any insulin-mediated effects on ventricular 6-phosphofructo-1-kinase activity and resultant effects on ventricular glycolysis do not appear to be exerted through changes in enzyme concentration, but probably through changes in modulators other than fructose-2,6-P2. In contrast to the ventricles, it appears that insulin exerts its effects on atrial 6-phosphofructo-1-kinase activity and, in part, influences atrial glycolysis through alteration of fructose-2,6-P2 levels, enzyme concentration, and isozymic content.     

Posttranslational modification of 6-phosphofructo-1-kinase in Aspergillus niger

Two different enzymes exhibiting 6-phosphofructo-1-kinase (PFK1) activity were isolated from the mycelium of Aspergillus niger: the native enzyme with a molecular mass of 85 kDa, which corresponded to the calculated molecular mass of the deduced amino acid sequence of the A. niger pfkA gene, and a shorter protein of approximately 49 kDa. A fragment of identical size also was obtained in vitro by the proteolytic digestion of the partially purified native PFK1 with proteinase K. When PFK1 activity was measured during the proteolytic degradation of the native protein, it was found to be lost after 1 h of incubation, but it was reestablished after induction of phosphorylation by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the system. By determining kinetic parameters, different ratios of activities measured at ATP concentrations of 0.1 and 1 mM were detected with fragmented PFK1, as with the native enzyme. Fructose-2,6-biphosphate significantly increased the Vmax of the fragmented protein, while it had virtually no effect on the native protein. The native enzyme could be purified only from the early stages of growth on a minimal medium, while the 49-kDa fragment appeared later and was activated at the time of a sudden change in the growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sequential purifications of PFK1 enzymes by affinity chromatography during the early stages of the fungal development suggested spontaneous posttranslational modification of the native PFK1 in A. niger cells, while from the kinetic parameters determined for both isolated forms it could be concluded that the fragmented enzyme might be more efficient under physiological conditions.     

Nature of the rat brain 6-phosphofructo-1-kinase isozymes

The complex nature of the brain 6-phosphofructo-1-kinase isozymes was examined by elution with a discontinuous gradient from QAE (quaternary aminoethyl)-Sephadex. In the first wash (150 mM NaCl), where the rat muscle 6-phosphofructo-1-kinase isozyme (M4) eluted, about 40% of the total brain 6-phosphofructo-1-kinase activity washed through without exhibiting a sharp peak. In the second elution (300 mM NaCl), the remaining activity eluted in a sharp peak that preceded where the major rat liver 6-phosphofructo-1-kinase isozyme (L4) eluted. Enzyme activity in brain extracts or purified brain isozymes was titrated above 90% with M4 anti-IgG and 20% with L4 anti-IgG. A purification procedure was developed which resulted in a recovery of 70 to 80% of the original enzyme activity in brain 100,000 X g supernatant fluids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on slab gels and detection by silver staining indicated that three components were present with apparent molecular weights of 87,500, 85,000, and 80,000. The 85,000- and 80,000-dalton components corresponded to the subunits of M4 and L4, respectively. The third component (C type) was thought to be an actual subunit since it exhibited the highest molecular weight and was present in an exhaustively washed immunoprecipitate of the purified brain isozymes. From 10 different purifications of the brain enzyme, the subunit distributions of the liver, muscle, and C-type subunit were 1.4 +/- 0.2, 4.9 +/- 0.5, and 3.9 +/- 0.3, respectively. A comparison of the kinetic properties of purified liver, muscle, and brain isozymes clearly demonstrated that all three preparations had quantitatively different regulatory properties. All three subunits were present in different regions of the brain, and region-specific changes in total activity and the relative amounts of each subunit were observed. This study suggests that brain 6-phosphofructo-1-kinase is a complex mixture of homotetramers and hybrids which are composed of different amounts of the three subunits.     

ATP and fructose-2,6-bisphosphate regulate skeletal muscle 6-phosphofructo-1-kinase by altering its quaternary structure

Recently, it has been demonstrated that fructose-2,6-bisphosphate (F2,6BP) protects skeletal muscle 6-phosphofructo-1-kinase (PFK) from thermal inactivation (50 degrees C) and against the deleterious effects of guanidinium hydrochloride (GdmCl). On the other hand, ATP, when added at its inhibitory concentrations, that is, >1 mM, enhanced either the thermal- or GdmCl-induced inactivation of PFK. Moreover, we concluded that these phenomena were probably due to the stabilization of PFK tetrameric structure by F2,6BP, and the dissociation of this structure into dimers induced by ATP. Aimed at elucidating the effects of F2,6BP and ATP on PFK at the structural and functional levels, the present work correlates the effects of these metabolites on the equilibrium between PFK dimers and tetramers to the regulation promoted on the enzyme catalytic activity. We show that ATP present a dual effect on PFK structure, favoring the formation of tetramer at stimulatory concentrations (up to 1 mM), and dissociating tetramers into dimers at inhibitory concentrations (>1 mM). Furthermore, F2,6BP counteracted this later ATP effect at either the structural or catalytic levels. Additionally, the effects of both F2,6BP or ATP on the equilibrium between PFK tetramers and dimers and on the enzyme activity presented a striking parallelism. Therefore, we concluded that modulation of PFK activity by ATP and F2,6BP is due to the effects of these ligands on PFK quaternary structure, altering the oligomeric equilibrium between PFK tetramers and dimers.     

Heart 6-phosphofructo-1-kinase. Subcellular distribution and binding to myofibrils

6-Phosphofructo-1-kinase (phosphofructokinase) (ATP:D-fructose-6-P 1-phosphotransferase, EC 2.7.1.11) can be identified in sheep heart homogenates in two forms, a soluble form and a form bound to the particulate fraction. Homogenates from immediately-dissected hearts have the enzyme in the soluble form, while those collected after a delay have the enzyme bound to the particulate fraction. Aldolase appears to show the same change in its location. Homogenization in a solution with concentrated macromolecular species (20% albumin) results in a greater association of phosphofructokinase and of aldolase to the particulate fraction in homogenates from immediately dissected hearts. Phosphofructokinase activity can be solubilized by two specific means: by high ionic strength, which is dependent upon specific salts; or by low ionic strength, which is dependent upon the presence of phosphofructokinase substrates or modifier ligands. These two means of solubilization are affected differently upon decreasing the pH below 6.9: the solubilization at low ionic strength is prevented, whereas phosphofructokinase is still solubilized by high ionic strength. Under the latter condition, the enzyme is in the inactive dimeric state, which can be activated at an alkaline pH. Myofibrils present in the particulate fraction can account for the binding of phosphofructokinase in heart homogenates. Purified myofibrils, when added to heart supernatant fluids, can bind phosphofructokinase at a slightly acidic pH. Conditions for phosphofructokinase binding to myofibrils, as well as its dissociation, follow what was observed with the binding of phosphofructokinase to the particulate fraction. At an acidic pH, and in the presence of a high concentration of ATP, phosphofructokinase exhibits low activity. However, if phosphofructokinase is assayed under these conditions while bound to myofibrils, the enzyme is activated.     

Evolution of allosteric citrate binding sites on 6-phosphofructo-1-kinase

As an important part of metabolism, metabolic flux through the glycolytic pathway is tightly regulated. The most complex control is exerted on 6-phosphofructo-1-kinase (PFK1) level; this control overrules the regulatory role of other allosteric enzymes. Among other effectors, citrate has been reported to play a vital role in the suppression of this enzyme's activity. In eukaryotes, amino acid residues forming the allosteric binding site for citrate are found both on the N- and the C-terminal region of the enzyme. These site has evolved from the phosphoenolpyruvate/ADP binding site of bacterial PFK1 due to the processes of duplication and tandem fusion of prokaryotic ancestor gene followed by the divergence of the catalytic and effector binding sites. Stricter inhibition of the PFK1 enzyme was needed during the evolution of multi-cellular organisms, and the most stringent control of PFK1 by citrate occurs in vertebrates. By substituting a single amino acid (K557R or K617A) as a component of the allosteric binding site in the C-terminal region of human muscle type PFK-M with a residue found in the corresponding site of a fungal enzyme, the inhibitory effect of citrate was attenuated. Moreover, the proteins carrying these single mutations enabled growth of E. coli transformants encoding mutated human PFK-M in a glucose-containing medium that did not support the growth of E. coli transformed with native human PFK-M. Substitution of another residue at the citrate-binding site (D591V) of human PFK-M resulted in the complete loss of activity. Detailed analyses revealed that the mutated PFK-M subunits formed dimers but were unable to associate into the active tetrameric holoenzyme. These results suggest that stricter control over glycolytic flux developed in metazoans, whose somatic cells are largely characterized by slow proliferation.     

citrate inhibition-resistant form of 6-phosphofructo-1-kinase from Aspergillus niger

Two forms of Aspergillus niger 6-phosphofructo-1-kinase (PFK1) have been described recently, the 85-kDa native enzyme and 49-kDa shorter fragment that is formed from the former by posttranslational modification. So far, kinetic characteristics have never been determined on the enzyme purified to near homogeneity. For the first time, kinetic parameters were determined for individual enzymes with respect to citrate inhibition. The native 85-kDa enzyme was found to be moderately inhibited by citrate, with the Ki value determined to be 1.5 mM, in the system with 5 mM Mg2+ ions, while increasing magnesium concentrations relieved the negative effect of citrate. An identical inhibition coefficient was determined also in the presence of ammonium ions, although ammonium acted as a strong activator of enzyme activity. On the other hand, the shorter fragment of PFK1 proved to be completely resistant to inhibition by citrate. Allosteric citrate binding sites were most probably lost after the truncation of the C-terminal part of the native protein, in which region some binding sites for inhibitor are known to be located. At near physiological conditions, characterized by low fructose-6-phosphate concentrations, a much higher efficiency of the shorter fragment was observed during an in vitro experiment. Since the enzyme became more susceptible to the positive control by specific ligands, while the negative control was lost after posttranslational modification, the shorter PFK1 fragment seems to be the enzyme most responsible for generating undisturbed metabolic flow through glycolysis in A. niger cells.     

Regulation of mammalian muscle type 6-phosphofructo-1-kinase and its implication for the control of the metabolism

Phosphofructokinase (PFK) is a major regulatory glycolytic enzyme and is considered to be the pacemaker of glycolysis. This enzyme presents a puzzling regulatory mechanism that is modulated by a large variety of metabolites, drugs, and intracellular proteins. To date, the mammalian enzyme structure has not yet been resolved. However, it is known that PFK undergoes an intricate oligomerization process, shifting among monomers, dimers, tetramers, and more complex oligomeric structures. The equilibrium between PFK dimers and tetramers is directly correlated with the enzyme regulation, because the dimer exhibits very low catalytic activity, whereas the tetramer is fully active. Several PFK ligands modulate the enzyme, favoring the formation of its dimers or tetramers. The present review integrates recent findings regarding the regulatory aspects of muscle type PFK and discusses their relation to the control of metabolism.     

Contribution of different protein phosphatases to the dephosphorylation of 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat liver.

The nature of rat liver protein phosphatases involved in the dephosphorylation of the glycolytic key enzyme 6-phosphofructo-1-kinase and the regulatory enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was investigated. In terms of the classification system proposed by Ingebritsen & Cohen [(1983) Eur. J. Biochem. 132, 255-261], only the type-2 protein phosphatases 2A (which can be separated into 2A1 and 2A2) and 2C act on these substrates. Fractionation of rat liver extracts by anion-exchange chromatography and gel filtration revealed that protein phosphatase 2A is responsible for most of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase phosphatase activity (activity ratio 2A/2C = 4:1). On the other hand, 6-phosphofructo-1-kinase phosphatase activity is equally distributed between protein phosphatases 2A (2A1 plus 2A2) and 2C. In addition, the possible role of low-Mr compounds for the control of purified protein phosphatase 2C was examined. At near-physiological concentrations, none of the metabolites studied significantly affected the rate of dephosphorylation of 6-phosphofructo-1-kinase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, pyruvate kinase or fructose-1,6-bisphosphatase.     

Regulation of 6-phosphofructo-1-kinase activity in ras-transformed rat-1 fibroblasts.

Fructose 2,6-bisphosphate (F-2,6-P2) stimulated glycolysis in cell-free extracts of both normal and ras-transfected rat-1 fibroblasts. The extract of the transformed cell glycolyzed more rapidly in both the absence and the presence of F-2,6-P2 than the extract of the parent fibroblast. Addition of mitochondrial ATPase (F1) or inorganic phosphate (Pi) further stimulated lactate production in both cell lines. F-2,6-P2 stimulated the 6-phosphofructo-1-kinase (PFK-1) activity in extracts of normal and transfected cells. The activity in extracts of transformed cells tested with a fructose 6-phosphate regenerating system was considerably higher than in the extract of normal cells. Stimulation of PFK-1 activity by cAMP of both cell lines was not as pronounced as that by F-2,6-P2. In the absence of F-2,6-P2 the PFK-1 activity was strongly inhibited in the transformed cell by ATP concentrations higher than 1 mM, whereas in the normal cell only a marginal inhibition was noted even at 2 or 3 mM ATP. F-2,6-P2 reversed the inhibition of PFK-1 by ATP. Nicotinamide adenine dinucleotide (NAD) at 100 microM (in the presence of 2 mM ATP and 1 microM F-2,6-P2) stimulated PFK-1 activity only in the transformed cell, whereas nicotinamide adenine dinucleotide phosphate (NADP) inhibited PFK-1 activity (in the presence or absence of 1 microM F-2,6-P2) in extracts of both cell lines. No previous observations of stimulation or inhibition by NAD or NADP on PFK-1 activity appear to have been reported. A threefold increase in the intracellular concentration of F-2,6-P2 was observed after transfection of rat-1 fibroblast by the ras oncogene. We conclude from these data that the PFK-1 activity of ras-transfected rat-1 fibroblasts shows a greater response to certain stimulating and inhibitory regulating factors than that of the parent cell.     

Alteration of 6-phosphofructo-1-kinase isozyme pools during heart development and aging

The nature of 6-phosphofructo-1-kinase isozyme pools in fetal, neonatal, young adult (3 months), and aged (30 months) rat hearts was studied using chromatographic and immunological techniques. Furthermore, the changing subunit composition of each isozyme pool was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 6% slab gels and by immunoblotting with subunit-specific antibodies. Although all three subunit types were expressed in heart throughout life, total activity and the nature of the isozyme pools varied during neonatal development and in aged heart. In fetal heart, the complex tetramers containing all three subunits appeared to be the major isozyme types. As the heart matured to the young adult stage, the M-type subunit increased over 6-fold; whereas the changes in the other two subunits were considerably less. These data indicate that during neonatal heart maturation the isozymic pools progressively exhibited increased amounts of the tetrameric forms containing two or more M-type subunits. In aged heart relative to the young adult (3 months) heart, the total activity and proportion of M-type subunit in the isozymes were decreased; and consequently, the amounts of the M-rich isozymes were decreased. The shifts in the types of isozymes during heart maturation and subsequent aging were primarily due to changes in availability of the M-type subunit to participate in random assembly of the tetrameric isozymes.     

Age-related changes in subunit composition and regulation of hepatic 6-phosphofructo-1-kinase.

6-Phosphofructo-1-kinase (PFK) isoenzyme pools from livers of fetal, neonatal, young adult (3 months) and aged (24 months) rats were studied. Near-term liver PFK isoenzyme pools were composed of nearly equal quantities of all three subunits. During the 30 days after birth, the total activity increased by 25%; the amount of the L-type, M-type or C-type subunit was increased 3-fold, was unchanged, or was decreased by 80% respectively. In aged rats, compared with young adults, total PFK activity was unchanged, but the L-type, M-type or C-type subunit decreased by 24%, increased by 39%, or increased by 338% respectively. During neonatal maturation, the changing subunit composition of the hepatic isoenzyme pools led to a decreased susceptibility to ATP inhibition, to a greater apparent affinity for fructose 6-phosphate, and to increased sensitivity to fructose 2,6-bisphosphate. Also, these alterations correlated with the measured increases in fructose 2,6-bisphosphate and the reported optimal rate of hepatic glycolysis/gluconeogenesis.     

Serotonin modulates hepatic 6-phosphofructo-1-kinase in an insulin synergistic manner

Human and rat hepatic tissue express many serotonin (5-HT) receptor subtypes, such as 5-HT_1B, 5-HT_2A, 5-HT_2B and 5-HT₇ receptors, which mediate diverse effects. 5-HT is known to regulate several key aspects of liver biology including hepatic blood flow, innervations and wound healing. 5-HT is also known to enhance net glucose uptake during glucose infusion in fasted dogs, but little is known about the ability of 5-HT to control hepatic glucose metabolism, especially glycolysis. This study addresses the potential of 5-HT to regulate PFK activity and the mechanisms related to the enzyme activity. Based on our results, we are the first to provide evidence that 5-HT up-regulates PFK in mouse hepatic tissue. Activation of the enzyme occurs through the 5-HT_2A receptor and phospholipase C (PLC), resulting in PFK intracellular redistribution and favoring PFK association to the cytoskeletal f-actin-enriched fractions. Interestingly, 5-HT and insulin act in a synergistic manner, likely because of the ability of insulin to increase fructose-2,6-bisphosphate because the presence of this PFK allosteric regulator enhances the 5-HT effect on the enzyme activity. Together, these data demonstrate the ability of 5-HT to control hepatic glycolysis and present clues about the mechanisms involved in these processes, which may be important in understanding the action of 5-HT during the hepatic wound healing process.     

Glucuronoxylomannan from Cryptococcus neoformans down-regulates the enzyme 6-phosphofructo-1-kinase of macrophages

The encapsulated yeast Cryptococcus neoformans is the causative agent of cryptococosis, an opportunistic life-threatening infection. C. neoformans is coated by a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is considered a key virulence factor of this pathogen. The present work aimed at evaluating the effects of GXM on the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK). GXM inhibited PFK activity in cultured murine macrophages in both dose- and time-dependent manners, which occurred in parallel to cell viability decrease. The polysaccharide also inhibited purified PFK, promoting a decrease on the enzyme affinity for its substrates. In macrophages GXM and PFK partially co-localized, suggesting that internalized polysaccharide directly may interact with this enzyme. The mechanism of PFK inhibition involved dissociation of tetramers into weakly active dimers, as revealed by fluorescence spectroscopy. Allosteric modulators of the enzyme able to stabilize its tetrameric conformation attenuated the inhibition promoted by GXM. Altogether, our results suggest that the mechanism of GXM-induced cell death involves the inhibition of the glycolytic flux.     

Phosphorylation and inactivation of yeast 6-phosphofructo-2-kinase contribute to the regulation of glycolysis under hypotonic stress.

Phosphorylation of yeast 6-phosphofructo-2-kinase and its role for the regulation of glycolysis under hypoosmotic conditions were investigated. 6-Phosphofructo-2-kinase was found to be phosphorylated in vitro by protein kinase C at serine 652 and thereby inactivated. Protein phosphatase 2A reversed the phosphorylative inhibition of the enzyme. When yeast cells were shifted to hypotonic media, 6-phosphofructo-2-kinase was found to be phosphorylated and inactivated. Under in vivo conditions, two phosphate residues were incorporated into the enzyme. One of them is bound to serine 652, indicating that this modification was probably caused by yeast protein kinase C1. The second phosphate is bound to Ser8 within the N-terminal peptide T(1-41) which contains several serine residues but no protein kinase C recognition sequence. Site-directed mutagenesis confirmed that the phosphorylation of serine 652 but not the N-terminal modification is responsible for the in vivo inactivation of 6-phosphofructo-2-kinase. The obtained results suggest that the phosphorylation of 6-phosphofructo-2-kinase mediates a response of the cells to an activation of the hypoosmolarity MAP kinase pathway. Via a suppression of glycolysis, the inactivation of 6-phosphofructo-2-kinase is expected to be responsible for the observed accumulation of glucose 6-phosphate, an essential precursor of the cell wall glucans, and the decrease of glycerol, an important osmolyte.     

Yeast 6-phosphofructo-2-kinase: sequence and mutant.

We have reported yeast 6-phosphofructo-2-kinase (EC 2.7.1.105) as having a ca. 96-kDa subunit size, as well as isolation of its structural gene, PFK26. Sequencing now shows an open reading frame of 827 amino acids and 93.5 kDa. The deduced amino acid sequence has 42% identity with the 55-kDa subunit of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver with extra material at both ends. Although the yeast sequence is especially similar to the liver one in its bisphosphatase domain, the essential His-258 of the liver enzyme is, in yeast, a serine, which may explain the apparent lack of bisphosphatase activity. Also, the yeast enzyme known to be activated via protein kinase A, has a putative phosphorylation site near its C-terminus and lacks the N-terminal phosphorylation sequence involved in inhibition of the liver enzyme. In a chromosomal null mutant strain, pfk26::LEU2, activity was marginal and the protein was not detectable as antigen. The mutant strain grew well on glucose and contained a near-normal level of fructose 2,6-P2. But in its growth on pyruvate, by contrast with the wild-type strain, no fructose 2,6-P2 was detectable, and it did not form after glucose addition in the presence of cycloheximide either. Such resting cells, however, metabolized glucose at the normal high rate. Glucose addition to the pfk26 mutant strain in the absence of cycloheximide, on the other hand, caused a ca. 10% normal rate of fructose 2,6-P2 accumulation, presumably employing a glucose-inducible second enzyme. Using strains also lacking 6-phosphofructo-1-kinase, affinity chromatography revealed the second enzyme as a minor peak amounting to 6% of 6-phosphofructo-2-kinase activity in a PFK26 strain and as the sole peak, in similar amount, in a pfk26 mutant strain.     

Metformin reverses hexokinase and 6-phosphofructo-1-kinase inhibition in skeletal muscle, liver and adipose tissues from streptozotocin-induced diabetic mouse

The present work describes the effects of metformin on hexokinase (HK) and phosphofructokinase (PFK) activities and localization in different tissues from streptozotocin-induced diabetic mice. Diabetic mice present lower HK and PFK activities (50%) in skeletal muscle, liver and adipose tissue, as compared with control (P < 0.05). Treatment with 250 mg/kg metformin reverses this pattern of enzyme inhibition with concomitant reversal of hyperglycemia and hypolactacidemia. Furthermore, the treatment increases the cytoskeleton-associated PFK activity in skeletal muscle; this activity has been described as an important mechanism for the enzyme activation. This effect might be due to the increased phosphorylation of serine residues in the enzyme, a modification which has been described to increase the interaction of PFK with f-actin. The current work supports the hypothesis that metformin hypoglycemic effects involve the activation of glycolysis through its regulatory enzymes, which may be potential targets for the development of new hypoglycemic drugs.