Chimeric phosphofructokinases involving exchange of the N- and C-terminal halves of mammalian isozymes: implications for ligand binding sites

Two phosphofructokinase (PFK) chimeras were constructed by exchanging the N- and C-terminal halves of the mammalian M- and C-type isozymes, to investigate the contribution of each terminus to the catalytic site and the fructose-2,6-P(2)/fructose-1,6-P(2) allosteric site. The homogeneously-purified chimeric enzymes organized into tetramers, and exhibited kinetic properties for fructose-6-P and MgATP similar to those of the native enzyme that furnished the N-terminal domain in each case, whereas their fructose-2,6-P(2) activatory characteristics coincided with those of the isozyme that provided the C-terminal half. This reflected the role of each domain in the formation of the corresponding binding site. Grafting the N-terminus of PFK-M onto the C-terminus of the fructose-1,6-P(2) insensitive PFK-C restored transduction of this signal to the catalytic site, which significance is also discussed.     

Phosphofructokinases from Escherichia coli. Purification and characterization of the nonallosteric isozyme.

The main phosphofructokinase of Escherichia coli (PFK I) is an extensively studied allosteric enzyme specified by the pfkA gene. A nonallosteric phosphofructokinase was reported (Fraenkel, D.G., Kotlarz, D., and Bluc, H. (1973) J. Biol. Chem. 248, 4865-4866) in strains carrying the pfkB1 mutation, a suppressor of pfkA mutants, and very low levels of this enzyme have also been detected in strains not carrying the suppressor (i.e. pfkB+). The nonallosteric protein has now been prepared pure from three strains, one carrying pfkB1 and pfkA+, one carrying pfkB1 and completely deleted for pfkA, and one carrying pfkB+ and also deleted for pfkA. It is apparently the same enzyme (PFK II) in all three strains, which shows that pfkB1 is a mutation affecting the amount of a normally minor isozyme. PFK II is a tetramer of slightly larger subunit molecular weight than PFK I (36,000 and 34,000, respectively). No immunological cross-reactivity was detected between PFK II and PFK I. Unlike PFK I, PFK II does not show cooperative interactions with fructose-6-P, inhibition by P-enolpyruvate, or activation by ADP. Also unlike PFK I, PFK II is somewhat sensitive to inhibition by fructose-1,6-P2 and can use tagatose-6-P as substrate. Both enzymes can perform the reverse reaction, fructose-6-P + ATP from fructose-1,6-P2 + ADP in vitro, but not in vivo. The normal function of PFK II is not known.     

腾冲嗜热厌氧菌6-磷酸果糖激酶的克隆、表达及其生物活性

6_磷酸果糖激酶(PFK)是糖酵解途径一个关键酶。基于腾冲嗜热厌氧菌基因组中的注释,基因TTE1816可能是PFK的一种,但是,它是否确有生物活性还必须有实验数据的支持。腾冲嗜热厌氧菌在最适温度培养后,提取细菌全蛋白,并采用双向电泳将可溶性蛋白质分离,然后运用质谱鉴定若干染色斑点。实验表明,TTE1816在高温条件下能够表达蛋白质。将TTE1816基因体外克隆至细菌表达载体,并在BL_21大肠杆菌中表达为可溶性蛋白。酶动力学实验表明,重组蛋白TTE1816具有PFK的催化活性,最适反应温度在60℃。它还能够催化葡萄糖、果糖、甘露糖和6_磷酸葡萄糖的磷酸化反应。另外,在高底物浓度和酶浓度的条件下,TTE1816还表现果糖二磷酸酶的特性。结果证明,TTE1816是腾冲嗜热厌氧菌中PFK家族的一个新成员。     

Purification and kinetic characterization of 6-phosphofructo-1-kinase from the liver of gilthead sea bream (Sparus aurata)

6-phosphofructo-1-kinase (PFK) was purified to homogeneity from liver of gilthead sea bream (Sparus aurata) and kinetic properties of the enzyme were determined. The native enzyme had an apparent molecular mass of 510 kDa and was composed of 86 kDa subunits, suggesting homohexameric structure. At pH 7, S. aurata liver PFK (PFKL) showed sigmoidal kinetics for fructose-6-phosphate (fru-6-P) and hyperbolic kinetics for ATP. Fructose-2,6-bisphosphate (fru-2,6-P2) converted saturation curves for fru-6-P to hyperbolic and activated PFKL synergistically with AMP. Fru-2,6-P2 counteracted the inhibition caused by ATP, ADP and citrate. Compared to the S. aurata muscle isozyme, PFKL had lower affinity for fru-6-P, higher cooperativity, hyperbolic kinetics in relation to ATP, increased susceptibility to inhibition by ATP, and was less affected by AMP, ADP and inhibition by 3-phosphoglycerate, phosphoenolpyruvate, 6-phosphogluconate or phosphocreatine. The effect of starvation-refeeding on PFKL expression was studied at the levels of enzyme activity and protein content in the liver of S. aurata. Our findings indicate that short-term recovery of PFKL activity after refeeding previously starved fish, may result from allosteric regulation by fru-2,6-P2, whereas combination of activation by fru-2,6-P2 and increase in protein content may determine the long-term recovery of the enzyme activity.     

Changes in the contents of metabolites and enzyme activities in rice plants responding to Rhizoctonia solani Kuhn infection: activation of glycolysis and connection to phenylpropanoid pathway

Rhizoctonia solani Kuhn causes sheath blight disease in rice, and genetic resistance against it is the most desirable characteristic. Current improvement efforts are based on analysis of polygenic quantitative trait loci (QTLs), but interpretation is limited by the lack of information on the changes in metabolic pathways. Our previous studies linked activation of the glycolytic pathway to enhanced generation of lignin in the phenylpropanoid pathway. The current studies investigated the regulation of glycolysis by examining the time course of changes in enzymatic activities and metabolite contents. The results showed that the activities of all glycolytic enzymes as well as fructose-6-phosphate (F-6-P), fructose-1,6-bisphosphate (F-1,6-P(2)), dihydroxyacetone phosphate (DHAP), glyceraldehyde-3-phosphate (GAP), 3-phosphoglycerate (3-PG), phosphoenolpyruvate (PEP) and pyruvate contents increased. These results combined with our previous findings that the expression of phosphoglucomutase (PGM), triosephosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase and pyruvate kinase (PK) increased after infection suggested that the additional establishment of glycolysis in the cytosol compartment occurred after infection. Further evidence for this was our recent findings that the increase in expression of the 6-phosphofructokinase (PFK) plastid isozyme Os06g05860 was accompanied by an increase in expression of three cytosolic PFK isozymes, i.e. Os01g09570, Os01g53680 and Os04g39420, as well as pyrophosphate-dependent phosphofrucokinase (PFP) isozymes Os08g25720 (α-subunit) and Os06g13810 (β-subunit) in infected rice plants of the resistant line. The results also showed that the reactions catalysed by PFK/PFP, aldolase, GAPDH + phosphoglycerate kinase (PGK) and PK in leaf sheaths of R. solani-infected rice plants were non-equilibrium reactions in vivo. This study showed that PGM, phosphoglucose isomerase (PGI), TPI and phosphoglycerate mutase (PGmu) + enolase could be regulated through coarse control whereas, PFK/PFP, aldolase, GAPDH + PGK and PK could be regulated through coarse and fine controls simultaneously.     

Regulation of glycolysis in sea bass liver: phosphofructokinase isozymes

Sea bass (Dicentrarchus labrax L.) liver phosphofructokinase (PFK) presents biphasic kinetics with respect to fructose-6-phosphate (F-6-P) in experiments carried out with crude extract. After the enzyme had been purified, two isozymes have been detected after chromatographic treatment. The two isozymes present different kinetic behaviour PFK-L1, the first eluted phosphofructokinase activity shows positive cooperativity with respect to fructose-6-phosphate and PFK-L2, the second activity fraction, has a Hill coefficient of 0.38 (negative cooperativity). The first isozyme shows less affinity for fructose-6-phosphate than that shown by PFK-L2. The joint kinetics of both isozymes produces a biphasic kinetics with respect to fructose-6-phosphate, similar to that observed in crude extracts.     

Accumulation of neutral lipids in Saccharomyces carlsbergensis by myo-inositol deficiency and its mechanism. Reciprocal regulation of yeast acetyl-CoA carboxylase by fructose bisphosphate and citrate.

The abnormal accumulation of lipids due to myo-inositol deficiency in Saccharomyces carlsbergensis, and the mechanism involved was investigated. The deficient cells contained much more neutral lipids with a greater ratio of unsaturated fatty acids compared to the supplemented cells, whereas there was no significant change in their phospholipid contents. The biosynthesis of fatty acids and sterols from acetate, and of triacylglycerols and sterol esters from palmitate was markedly augmented in the deficient cells. Acetyl-CoA carboxylase activity of the deficient supernatant was 2- to 5-fold higher than that of the supplemented. However, the activity from both sources was not significantly different after Sephadex G-25 gel filtration of the supernatant, suggesting the presence of low molecular effector(s) in the deficient supernatant. There was a great increase in acid-soluble glycogen, trehalose, and fructose-1,6-P2, as well as a drastic decrease in citrate in the deficient cells. Their intracellular levels were calculated so that their effects on acetyl-CoA carboxylase was examined over the range of physiological concentration. Citrate strongly inhibited the enzyme activity of the supernatant, but it had no effect on the preparation after gel filtration. On the other hand, fructose-1,6-P2 stimulated the enzyme activity both before and after gel filtration. The acetyl-CoA carboxylase activity in the gel filtrate was measured as a function of citrate concentration at several fixed concentrations of fructose-1,6-P2. Citrate counteracted the activation by fructose-1,6-P2 in a dose-dependent manner. Citrate lacked the inhibitory effect in the absence of fructose-1,6-P2. It was concluded from these results that neutral lipid accumulation in the deficient cells reflected an increase in the synthesis of fatty acids, at least partly based on an enhancement of acetyl-CoA carboxylase activity, and that the operation of a reciprocal regulation of the enzyme by fructose-1,6-P2 and citrate caused a marked elevation of the enzyme activity in the deficient cells with a high fructose-1,6-P2 level and a low citrate level.     

Sucrose metabolism in developing endosperm of immature wheat (Triticum aestivum L.) grain

With a view to investigating the role of the enzyme pyrophosphate-fructose-6-phosphate-1-phosphotransferase (PFP) in sucrose breakdown in developing endosperm of wheat grain, the activity of PFP and related enzymes such as phosphofructokinase (PFK), fructose-6-bisphosphatase (FBPase), fructose-6-phosphate-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (F2, 6-P2ase) and the contents of the various intermediates of the pathway serving either the substrate or the effectors of these enzymes such as glu-6-P,glu-1-P,fru-6-P,fru-1,6-P2,DHAP,G3P, UDP-glucose, ADP-glucose, Pi,PPi and fru-2,6-P2 have been determined at 5 days intervals starting from day-5 after anthesis until day-40 after anthesis. These enzymes except PFK-2 had their peak activity at day-25 after anthesis. The activity of PFP was several fold higher than that of PFK at each stage of grain development. PFK-2 exhibited the lowest activity. The various intermediates again had their maximum concentration either at day-20 or day-25 after anthesis. Among hexose phosphates studied, glu-6-P was present in highest concentration at each stage of grain development. The level of Pi was much higher than those of PPi and fru-2,6-P2. Similarly, concentration of UDP-glucose was higher than that of ADP-glucose. Based on these results, it is proposed that the major role of the enzyme PFP in developing wheat grain is to provide PPi for sucrose breakdown via sucrose synthase.     

Phosphofructokinase isozyme expression during myoblast differentiation

Isozyme expression of phosphofructokinase (PFK), the key regulatory enzyme for glycolysis, was studied during differentiation of mouse C2 myoblasts to myotubes. The total PFK activity increased 20-fold during in vitro myogenesis. The rate of synthesis, relative to the rate of total protein synthesis, measured by pulse labeling and immunoprecipitation was lowest for muscle PFK (PFK-A), 0.008% in myoblasts, while those for liver (PFK-B) and brain (PFK-C) PFK were 0.017 and 0.014%, respectively. The relative rate of PFK-A synthesis increased sharply (5-fold) at an initial period of differentiation (8 h) and reached maximum of 10-fold at 48 h, to make PFK-A the major isoform synthesized in myotubes. The relative rates of synthesis for both PFK-B and PFK-C did not change drastically, decreasing slightly at 8 h, but were restored to 1.5-2-fold of myoblasts. cDNA sequences coding for mouse muscle PFK were cloned and used along with those for mouse liver PFK, which we have previously cloned, to measure by Northern blot analysis under highly stringent conditions the steady-state mRNA concentrations for muscle and liver PFK during C2 differentiation. The hybridizable mRNA level for PFK-A increased gradually, reaching 13-fold at 48 h when 80% of cells was fused to myotubes. The PFK-A mRNA level at 96 h was 90-fold of that for myoblasts. In contrast, the mRNA level for PFK-B increased slightly during differentiation, showing a maximum of 4-fold at 96 h. These results indicate isozyme-specific control of muscle PFK gene expression during C2 myoblast differentiation.     

Biochemical studies of canine muscle phosphofructokinase deficiency

Skeletal muscle from four dogs with erythrocyte phosphofructokinase (PFK; EC 2.7.1.11) deficiency were studied in vitro. Muscle PFK activities were severely decreased to 1% of the normal mean. The residual activities had a high Km for fructose-6-phosphate (F6P). Anaerobic lactate production of PFK-deficient muscle was minimal with the addition of glycogen and hexose-monophosphates, but was normal with fructose-1,6-diphosphate (FDP). Muscle glycogen concentration was twice normal, indicating a glycogen storage disorder. Histochemical studies for muscle PFK activity showed no enzymatic staining with F6P as substrate. In two muscle biopsies from asymptomatic related dogs, intermediate PFK activities were found. These data characterize canine muscle PFK deficiency in vitro.     

Activation of muscle phosphofructokinase by fructose 2,6-bisphosphate and fructose 1,6-bisphosphate is differently affected by other regulatory metabolites

Fructose-2,6-P2 and fructose-1,6-P2 are strong activators of muscle phosphofructokinase. They have been shown to be competitive in binding studies, and it is generally thought that they affect the physical and catalytic properties of the enzyme in the same manner. However, there are indications in published data that the effects of the two fructose bisphosphates on phosphofructokinase are not identical. To examine this possibility, the kinetics of activation of rat skeletal muscle phosphofructokinase by the two fructose bisphosphates were compared in the presence of other regulatory metabolites. Citrate greatly increased the K0.5 of the enzyme for fructose-2,6-P2, with little effect on the maximum activation. In contrast, citrate greatly decreased the maximum activation by fructose-1,6-P2, with only a small effect on the K0.5. Changes in the concentrations of the inhibitor ATP or the activator AMP similarly altered the K0.5 for fructose-2,6-P2, but altered the maximum activation by fructose-1,6-P2. Finally, when fructose-1,6-P2 was added in the presence of a given concentration of fructose-2,6-P2, phosphofructokinase activity was decreased if the activation by fructose-2,6-P2 alone was greater than the maximum activation by fructose-1,6-P2 alone. These results are consistent with competition of the two fructose bisphosphates for the same binding site, but indicate that the conformational changes produced by their binding are different.     

Contributions of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 to the elevated glycolysis in hepatocytes from Zucker fa/fa rats

The insulin-resistant Zucker fa/fa rat has elevated hepatic glycolysis and activities of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 (PFK2). The latter catalyzes the formation and degradation of fructose-2,6-bisphosphate (fructose-2,6-P(2)) and is a glucokinase-binding protein. The contributions of glucokinase and PFK2 to the elevated glycolysis in fa/fa hepatocytes were determined by overexpressing these enzymes individually or in combination. Metabolic control analysis was used to determine enzyme coefficients on glycolysis and metabolite concentrations. Glucokinase had a high control coefficient on glycolysis in all hormonal conditions tested, whereas PFK2 had significant control only in the presence of glucagon, which phosphorylates PFK2 and suppresses glycolysis. Despite the high control strength of glucokinase, the elevated glycolysis in fa/fa hepatocytes could not be explained by the elevated glucokinase activity alone. In hepatocytes from fa/fa rats, glucokinase translocation between the nucleus and the cytoplasm was refractory to glucose but responsive to glucagon. Expression of a kinase-active PFK2 variant reversed the glucagon effect on glucokinase translocation and glucose phosphorylation, confirming the role for PFK2 in sequestering glucokinase in the cytoplasm. Glucokinase had a high control on glucose-6-phosphate content; however, like PFK2, it had a relative modest effect on the fructose-2,6-P(2) content. However, combined overexpression of glucokinase and PFK2 had a synergistic effect on fructose-2,6-P(2) levels, suggesting that interaction of these enzymes may be a prerequisite for formation of fructose-2,6-P(2). Cumulatively, this study provides support for coordinate roles for glucokinase and PFK2 in the elevated hepatic glycolysis in fa/fa rats.     

Comparison of the Activities and Some Properties of Pyrophosphate and ATP Dependent Fructose-6-Phosphate 1-Phosphotransferases of Phaseolus vulgaris Seeds

The distribution of pyrophosphate: fructose 6-phosphate phosphotransferase (PFP) and ATP: fructose-6-phosphate 1-phosphotransferase (PFK) was studied in germinating bean (Phaseolus vulgaris cv Top Crop) seeds. In the cotyledons the PFP activity was comparable with that of PFK. However, in the plumule and radicle plus hypocotyl, PFP activity exceeds that of PFK. Approximately 70 to 90%, depending on the stage of germination, of the total PFP and PFK activities were present in the cotyledons. Highest specific activity of both enzymes, however, occurred in the radicle plus hypocotyl (64-90 nanomoles·min·milligram protein). Fractionation studies indicate that 40% of the total PFK activity was associated with the plastids while PFP is apparently confined to the cytoplasm. The cytosolic isozyme of PFK exhibits hyperbolic kinetics with respect to fructose 6-P and ATP with K_m values of 320 and 46 micromolar, respectively. PFP also exhibits hyperbolic kinetics both in the presence and absence of the activator fructose-2,6-P₂. The activation is caused by lowering the K_m for fructose 6-P from 18 to 1.1 millimolar and that for pyrophosphate (PPi) from 40 to 25 micromolar, respectively. Levels of fructose 2,6-P₂ and PPi in the seeds are sufficient to activate PFP and thereby enable a glycolytic role for PFP during germination. However, the fructose 6-P content appears to be well below the K_m of PFP for this compound and would therefore preferentially bind to the catalytic site of PFK, which has a lower K_m for fructose 6-P. The ATP content appears to be at saturating levels for PFK.     

Expression of key substrate cycle enzymes in rat spermatogenic cells: fructose 1,6 bisphosphatase and 6 phosphofructose 1-kinase

A substrate cycle composed of phosphofructo 1-kinase I (PFK) and fructose 1,6 bisphosphatase I (FBPase) has been proposed in rat spermatids. This substrate cycle can explain the ability of glucose to induce a decrease in intracellular ATP, a phenomenon that was related to regulation of [Ca(2+)]i in these cells. In spite of the importance of this metabolic cycle, the expression and activities of the enzymes that compose such cycle have not been systematically studied in spermatogenic cells. Here, we show that PFK and FBPase activities were present in pachytene spermatocytes and round spermatids extracts. Expression of PFK at the mRNA and protein levels showed a relatively similar expression in spermatogenic cells, but a stronger expression in Sertoli cells. Instead, expression of FBPase at the mRNA and protein levels was stronger in round and elongating spermatids as compared to other spermatogenic cells. A similar pattern was observed when evidencing FBPase activity by a NADPH-nitroblue tetrazolium-linked cytochemical assay in isolated pachytene spermatocytes and round spermatids. Rat spermatids also showed the ability to convert lactate to fructose- and glucose-6-P, indicating that both glycolytic and gluconeogenic fluxes are present in these cells. Our results indicate that a coordinated expression of key substrate cycle enzymes, at the level of PFK/FBPase, appear in the last stages of spermatogenic cell differentiation, suggesting that the co-regulation of these enzymes are required for the ability of these cells to respond to glucose and induce metabolic and Ca(2+) signals that can be important for sperm development and function.     

Mechanism of action of 2,5-anhydro-D-mannitol in hepatocytes. Effects of phosphorylated metabolites on enzymes of carbohydrate metabolism

Isolated rat hepatocytes convert 2,5-anhydromannitol to 2,5-anhydromannitol-1-P and 2,5-anhydromannitol-1,6-P2. Cellular concentrations of the monophosphate and bisphosphate are proportional to the concentration of 2,5-anhydromannitol and are decreased by gluconeogenic substrates but not by glucose. Rat liver phosphofructokinase-1 phosphorylates 2,5-anhydromannitol-1-P; the rate is less than that for fructose-6-P but is stimulated by fructose-2,6-P2. At 1 mM fructose-6-P, bisphosphate compounds activate rat liver phosphofructokinase-1 in the following order of effectiveness: fructose-2,6-P2 much greater than 2,5-anhydromannitol-1,6-P2 greater than fructose-1,6-P2 greater than 2,5-anhydroglucitol-1,6-P2. High concentrations of fructose-1,6-P2 or 2,5-anhydromannitol-1,6-P2 inhibit phosphofructokinase-1. Rat liver fructose 1,6-bisphosphatase is inhibited competitively by 2,5-anhydromannitol-1,6-P2 and noncompetitively by 2,5-anhydroglucitol-1,6-P2. The AMP inhibition of fructose 1,6-bisphosphatase is potentiated by 2,5-anhydroglucitol-1,6-P2 but not by 2,5-anhydromannitol-1,6-P2. Rat liver pyruvate kinase is stimulated by micromolar concentrations of 2,5-anhydromannitol-1,6-P2; the maximal activation is the same as for fructose-1,6-P2. 2,5-Anhydroglucitol-1,6-P2 is a weak activator. 2,5-Anhydromannitol-1-P stimulates pyruvate kinase more effectively than fructose-1-P. Effects of glucagon on pyruvate kinase are not altered by prior treatment of hepatocytes with 2,5-anhydromannitol. Pyruvate kinase from glucagon-treated hepatocytes has the same activity as the control pyruvate kinase at saturating concentrations of 2,5-anhydromannitol-1,6-P2 but has a decreased affinity for 2,5-anhydromannitol-1,6-P2 and is not stimulated by 2,5-anhydromannitol-1-P. The inhibition of gluconeogenesis and enhancement of glycolysis from gluconeogenic precursors in hepatocytes treated with 2,5-anhydromannitol can be explained by an inhibition of fructose 1,6-bisphosphatase, an activation of pyruvate kinase, and an abolition of the influence of phosphorylation on pyruvate kinase.     

Purification and characterization of phosphofructokinase of Bacillus licheniformis

The phosphofructokinase (PFK) of Bacillus licheniformis was purified about 50–65-fold and examined for a number of enzymatic and physical characteristics. The enzyme is quite unstable under normal assay conditions, but Mg²⁺, K⁺, adenosine-5′-diphosphate, phosphoenolpyruvate (PEP), and fructose-6-phosphate (fru-6-P) are fairly effective stabilizing agents. Saturation functions for ATP and fru-6-P were hyperbolic. Several attempts to induce positive cooperative binding of fru-6-P were unsuccessful. However, “sigmoidal” saturation kinetics for fru-6-P could be observed under assay conditions that permitted an irreversible inactivation of the PFK during assay. Several divalent cations could support the catalysis of B. licheniformis PFK and the enzyme was activated by both NH₄⁺ and K⁺ ions. B. licheniformis PFK is inhibited by citrate, ATP, PEP, Ca²⁺, and several other metabolic intermediates, but the inhibition caused by citrate and ATP at high fru-6-P concentration and by calcium can be relieved by Mg²⁺ addition while PEP inhibition is specifically relieved by fru-6-P. There are at least three binding sites for PEP on the PFK molecule. The active form of this PFK has a molecular weight of about 134,000 daltons. In the presence of Mg²⁺, adenosine-5′-triphosphate (ATP), and PEP, at 0 °C, the PFK molecule is rapidly dissociated to an inactive form with a molecular weight of about 68,000 daltons. Association of these subunits to yield the active form of PFK occurs spontaneously, and rapidly, when the temperature is raised to 30 °C. Ninety percent of the original activity is recovered after activation. Growth of B. licheniformis on several different substrates resulted in minor variations of PFK activity. In a parallel fashion, sporulation involved no irreversible inactivation of PFK and the level of the activity was about the same throughout the life cycle. Control of this enzyme during sporulation could be affected by any or all of the cell constituents found to regulate PFK activity in vitro, but it is considered likely that the most significant in vivo negative effector is PEP, with this inhibition being reversed by fru-6-P.     

Oscillatory synthesis of glucose 1,6-bisphosphate and frequency modulation of glycolytic oscillations in skeletal muscle extracts

Oscillatory behavior of glycolysis in cell-free extracts of rat skeletal muscle involves bursts of phosphofructokinase activity, due to autocatalytic activation by fructose-1,6-P2. Glucose-1,6-P2 similarly might activate phosphofructokinase in an autocatalytic manner, because it is produced in a side reaction of phosphofructokinase and in a side reaction of phosphoglucomutase using fructose-1,6-P2. When muscle extracts were provided with 1 mM ATP and 10 mM glucose, glucose-1,6-P2 accumulated in a stepwise, but monotonic, manner to 0.7 microM in 1 h. The stepwise increases occurred during the phases when fructose-1,6-P2 was available, consistent with glucose-1,6-P2 synthesis in the phosphoglucomutase side reaction. Addition of 5-20 microM glucose-1,6-P2 increased the frequency of the oscillations in a dose-dependent manner and progressively shortened the time interval before the first burst of phosphofructokinase activity. Addition of 30 microM glucose-1,6-P2 blocked the oscillations. The peak values of the [ATP]/[ADP] ratio were then eliminated, and the average [ATP]/[ADP] ratio was reduced by half. In the presence of higher, near physiological concentrations of ATP and citrate (which reduce the activation of phosphofructokinase by glucose-1,6-P2), high physiological concentrations of glucose-1,6-P2 (50-100 microM) increased the frequency of the oscillations and did not block them. We conclude that autocatalytic activation of phosphofructokinase by fructose-1,6-P2, but not by glucose-1,6-P2, is the mechanism generating the oscillations in muscle extracts. Glucose-1,6-P2 may nevertheless play a role in facilitating the initiation of the oscillations and in modulating their frequency.     

Mechanisms of glycolytic control during hibernation in the ground squirrel Spermophilus lateralis

Summary The mechanisms of glycolytic rate control during hibernation in the ground squirrel Spermophilus lateralis were investigated in four tissues: heart, liver, kidney, and leg muscle. Overall glycogen phosphorylase activity decreased significantly in liver and kidney to give 50% or 75% of the activity found in the corresponding euthermic organs, respectively. The concentration of fructose-2,6-bisphosphate (F-2,6-P₂) decreased significantly in heart and leg muscle during hibernation to 50% and 80% of euthermic tissue concentrations, respectively, but remained constant in liver and kidney. The overall activity of pyruvate dehydrogenase (PDH) in heart and kidney from hibernators was only 4% of the corresponding euthermic values. Measurements of phosphofructokinase (PFK) and pyruvate kinase (PK) kinetic parameters in euthermic and hibernating animals showed that heart and skeletal muscle had typical rabbit skeletal M-type PFK and M₁-type PK. Liver and kidney PFK were similar to the L-type enzyme from rabbit liver, whereas liver and kidney PK were similar to the M₂ isozyme found primarily in rabbit kidney. The kinetic parameters of PFK and PK from euthermic vs hibernating animals were not statistically different. These data indicate that tissue-specific phosphorylation of glycogen phosphorylase and PDH, as well as changes in the concentration of F-2,6-P₂ may be part of a general mechanism to coordinate glycolytic rate reduction in hibernating S. lateralis.Abbreviations ADP adenosine diphosphate - AMP adenosine monophosphate - ATP adenonine triphoshate - EDTA ethylenediaminetetra-acetic acid - EGTA ethylene glycol tetra-acetic acid - F-6-P fructose 6-phosphate - F-1,6-P₂ fructose 1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate - K _a activation coefficient - I₅₀ concentration of inhibitor which reduces control activity by 50% - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PFK 6-phosphofructo-1-kinase - PK pyruvate kinase     

Physiological relevance of the changing subunit composition and regulatory properties of the 6-phosphofructo-1-kinase isozyme pools during heart and muscle development

During postnatal development, the subunit compositions of the 6-phosphofructo-l-kinase isozyme pools of heart and skeletal muscle are known to change. The isozyme pools from fetal muscle were composed of the L-type (60%), and M-type (36%) and C-type (4%) subunits and the isozymes from fetal and early neonatal heart contain nearly equal amounts of all three subunits. During postnatal development of both tissues, the proportion of the M-type subunit increases until it is the only type present in adult muscle and the major subunit in adult heart (7507o). The isozyme pool from fetal muscle exhibit a decreased affinity for fructose-6-P and a greater susceptibility to ATP inhibition compared to the M-rich isozymes which are subsequently present. The isozyme pools from fetal and early neonatal heart, if compared to the M-rich isozymes which are present later during heart development and to the fetal muscle isozymes, exhibited the least affinity for fructose-6-P and the greatest susceptibility to ATP inhibition. Comparison of the isozyme pools containing little or no C-type subunit with those from fetal and early neonatal heart clearly indicates that the presence of substantial levels of the C-type subunit imposed a decreased ability for fructose-2,6-P₂ to both lower affinity for fructose-6-P and antagonize sensitivity to ATP inhibition. Although still not thoroughly appreciated, it appears that the changing nature of the isozyme pools in these tissues permits regulation of glucose metabolism in a manner which allows efficient utilization of nutritional opportunities and which adequately meets the energy requirements of each tissue at different stages of development.Abbreviations PFK 6-phosphofructo-l-kinase - fructose-6-P D-fructose-6-phosphate - fr-t_ose-2,6-P₂ D-fructose-2,6-bisphosphate     

The role of phosphofructokinase in glycolytic control in the facultative anaerobe Sipunculus nudus

Summary The involvement of phosphofructokinase (PFK) in glycolytic control was investigated in the marine peanut worm Sipunculus nudus. Different glycolytic rates prevailed at rest and during functional and environmental anaerobiosis: in active animals glycogen depletion was enhanced by a factor of 120; during hypoxic exposure the glycolytic flux increased only slightly. Determination of the mass action ratio (MAR) revealed PFK as a non-equilibrium enzyme in all three physiological situations. Duirng muscular activity the PFK reaction was shifted towards equilibrium; this might account for the observed increase in glycolytic rate under these conditions. PFK was purified from the body wall muscle of S. nudus. The enzyme was inhibited by physiological ATP concentrations and an acidic pH; adenosine monophosphate (AMP), inorganic phosphate (P_i), and fructose-2,6-bisphosphate (F-2,6-P₂) served as activators. PFK activity, determined under simulated cellular conditions of rest and muscular work, agreed well with the glycolytic flux in the respective situations. However, under hypoxia PFK activity surpassed the glycolytic rate, indicating that PFK may not be rate-limiting under these conditions. The results suggest that glycolytic rate in S. nudus is mainly regulated by PFK during rest and activity. Under hypoxic conditions the regulatory function of PFK is less pronounced.Abbreviations ATP, ADP, AMP adenosine tri-, di-, monophosphate - DTT dithiothreitol - EDTA ethylene diaminetetra-acetic acid - F-6-P fructose-6-phosphate - F-1,6-P₂ fructose-1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate; bwm, body wall muscle; fresh mass, total body weight - G-6-P glucose-6-phosphate - H enthalpy change - K _a activation constant - K _eq equilibrium constant - K _i inhibition constant - K _m Michaelis constant - MAR mass action ratio - NMR nuclear magnetic resonance - PFK phosphofructokinase - P_i inorganic phosphate - PLA phospho-l-arginine - SD standard deviation - TRIS, TRIS (hydroxymethyl) aminomethane - TRA triethanolamine hydrochloride - V _max maximal velocity