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PubMed1994

Allosteric activation in Bacillus stearothermophilus lactate dehydrogenase investigated by an X-ray crystallographic analysis of a mutant designed to prevent tetramerization of the enzyme.

The crystal structure of a mutant Bacillus stearothermophilus lactate dehydrogenase, into which an additional loop has been engineered in order to prevent tetramerization of the enzyme, has been solved and refined at 2.4 A. The minimal repeat unit in the crystal is a dimer and the tetramer cannot be generated by any of the crystallographic symmetry operations in P2(1). The loop protrudes out into the solvent, stabilized by a good hydrogen bonding arrangement, and clearly sterically hinders tetramer formation. This is the first structure of B. stearothermophilus lactate dehydrogenase (bsLDH) in which the allosteric activator fructose, 1,6-bisphosphate (FBP) is not present. To investigate the mechanism of allosteric activation in this enzyme we have compared the structure with a ternary complex of B. stearothermophilus lactate dehydrogenase. Many of our observations confirm those reported from a comparison of FBP-bound ternary bsLDH complex with an FBP free LDH from another bacterial source, Bifidobacterium longum. Our results suggest that quaternary structural alterations may have less influence on the mechanism than previously reported. The differences in the quaternary structural behaviour of these two enzymes is discussed.

Allosteric Regulation↗
PubMed1993

A 31P NMR spectroscopy study of Xenopus laevis heart perfused in vitro with creatinol-O-phosphate, phosphocreatine, adenosine triphosphate, fructose diphosphate and ouabain.

Xenopus laevis heart was studied by 31P NMR using a 200 MHz proton spectrometer; hearts were perfused, at pH 7.35 and room temperature, with normal oxygenated or K(+)-enriched Ringer. Solution was later added with creatinol-O-phosphate (COP), phosphocreatine (PCr), adenosine triphosphate (ATP), fructose-1,6-diphosphate (FDP) and ouabain. NMR spectra of the heart show organic phosphomono- and phosphodi-esters, inorganic phosphate, PCr, overlapping alpha-ATP/ADP and gamma-ATP/beta-ADP, and beta-ATP signals. Their chemical shift positions and areas showed no significant changes in the course of 1.5 h perfusions with either solution, except in a few preparations, whether the heart was beating or reversibly arrested. While COP reduced the signals in beating hearts, the same spectra exhibited no consistent, substantial changes under PCr, ATP and FDP 1 to 10 mM, pH 7.35 perfusion with either solution, nor when ouabain mumol was added. The spectra are briefly discussed in comparison with those observed in the perfused heart of mammals (mostly rat), and particularly with those obtained in the frog (Rana temporaria) heart, both by analysing the bioenergetic equilibria on the basis of total tissue substrate levels measured in extracts of freeze-clamped tissue, and by evaluating cytochrome-b, flavin and pyridine nucleotide in vitro oxido-reduction read-outs in separate, similar experimental settings.

Adenosine Triphosphate↗
PubMed1988

Cardiac metabolism as an indicator of oxygen supply/demand ratio.

We evaluated the anti-ischemic effect of drugs by using the inhibition of glycolytic flux at the level of the phosphofructokinase (PFK) reaction, caused by ischemia, as an indicator of the oxygen supply/demand ratio in the ischemic myocardium. Ischemia was induced by ligating the left anterior descending coronary artery in the open-chest dog. After 3 min of coronary ligation, the ischemic myocardium was removed. The endocardial portion of the myocardial sample was used to determine the levels of glucose-6-phosphate (G6P), fructose-6-phosphate (F6P) and fructose-1,6-diphosphate (FDP), and the ratio of [( G6P] + [F6P])/[FDP] was calculated in order to assess the rate of glycolytic flux at the PFK stage. Either saline or drug (propranolol, 1 mg/kg; carteolol, 100 micrograms/kg; nadolol, 1 mg/kg; nifedipine, 10 micrograms/kg; diltiazem, 100 micrograms/kg; verapamil, 100 micrograms/kg; and flunarizine, 1 mg/kg) was injected intravenously 5 min before coronary ligation. In the saline-treated heart, ischemia increased the levels of G6P and F6P, whereas it decreased the level of FDP. The ratio of ([G6P] + [F6P])/[FDP] was increased by ischemia from 2.2 to 23.6, suggesting the inhibition of glycolytic flux at the level of the PFK reaction. In the drug-treated heart, ischemia increased the levels of G6P and F6P, but the increases were smaller than those in the saline-treated heart. Pretreatment with propranolol, nadolol, diltiazem, verapamil, flunarizine attenuated the increase in the ratio of ([G6P] + [F6P])/[FDP] caused by ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Role of timing of administration in the cardioprotective effect of fructose-1,6-bisphosphate.

We administered fructose-1,6-bisphosphate (FDP), 1 mM, to isolated and perfused rabbit hearts submitted, after 90 minutes of equilibration, to an ischemic period (60 minutes at a coronary flow of 0.17 ml/min/g), followed by a period of reperfusion (30 minutes at a coronary flow of 3.6 ml/min/g). FDP was delivered at different times following the experimental protocol: 60 minutes before ischemia and for the entire experiment; 60 minutes before and during ischemia, but not at reperfusion; at the onset of ischemia and during reperfusion; and only during reperfusion. The FDP cardioprotective effect was evaluated in terms of recovery of left ventricular pressure developed during reperfusion, creatine phosphokinase (CPK) and noradrenaline release, mitochondrial function (expressed as yield, RCI, QO2, ADP/O), ATP and creatine phosphate (CP) tissue contents, calcium homeostasis, and by measuring oxidative stress in terms of reduced and oxidized glutathione release and tissue contents. Our data show that the cytoprotective action of FDP is closely related to the time of administration. Optimal myocardial preservation was achieved when it was present prior to ischemia and during reperfusion. When given at the time of ischemia or only on reperfusion, FDP does not exert cardioprotection. The data suggest that the FDP cardioprotective effect is related to improvement of energy metabolism.

Adenosine Triphosphate↗
PubMed1990

Fructose-1,6-diphosphate counteracts ethanol-stimulated calcium uptake in isolated BHK cells.

Ethanol increases the uptake of 45Ca by isolated baby hamster kidney (BHK) cells in vitro. The effect is dependent on ethanol and 45Ca++ concentration and on the incubation time. Fructose-1,6-diphosphate (FDP) added at different concentration during the pre-incubation exerts a protective effect through a membrane-stabilizing action which is consistent with its in vivo anti-alcohol activity documented in previous studies.

Animals↗
PubMed1982

Fructose-2,6-P2, chemistry and biological function.

A new activator of phosphofructokinase, which is bound to the enzyme and released during its purification, has been discovered. Its structure has been determined as beta-D Fructose-2,6-P2 by chemical synthesis, analysis of various degradation products and NMR. D-Fructose-2,6-P2 is the most potent activator of phosphofructokinase and relieves inhibition of the enzyme by ATP and citrate. It lowers the Km for fructose-6-P from 6 mM to 0.1 mM. Fructose-6-P,2-kinase catalyzes the synthesis of fructose-2,6-P2 from fructose-6-P and ATP, and the enzyme has been partially purified. The degradation of fructose-2,6-P2 is catalyzed by fructose-2,6-bisphosphatase. Thus a metabolic cycle could occur between fructose-6-P and fructose-2,6-P2, which are catalyzed by these two opposing enzymes. The activities of these enzymes can be controlled by phosphorylation. Fructose-6-P,2-kinase is inactivated by phosphorylation catalyzed by either cAMP dependent protein kinase or phosphorylase kinase. The inactive, phospho-fructose-6,P,2-kinase is activated by dephosphorylation catalyzed by phosphorylase phosphatase. On the other hand, fructose-2,6-bisphosphatase is activated by phosphorylation catalyzed by cAMP dependent protein kinase. Investigation into the hormonal regulation of phosphofructokinase reveals that glucagon stimulates phosphorylation of phosphofructokinase which results in decreased affinity for fructose-2,6-P2 appears to be due to the decreased synthesis by inactivation of fructose-2,6-P2,2-kinase and increased degradation as a result of activation of fructose-2,6-bisphosphatase. Such a reciprocal change in these two enzymes has been demonstrated in the hepatocytes treated by glucagon and epinephrine. The implications of these observations in respect to possible coordinated controls of glycolysis and glycogen metabolism are discussed.

Enzyme Activation↗
PubMed1992

A kinetic description of sequential, reversible, Michaelis-Menten reactions: practical application of theory to metabolic pathways.

Equations are presented which describe a linear coupled system of reactions that utilize a single substrate and convert it to product by way of several intermediate enzyme catalysed steps. The present analysis extends previous results by assuming that the enzymes obey reversible Michaelis-Menten kinetics. In order for the system to reach steady state one must assume that the initial substrate concentration and the final product concentration are buffered to a constant value. Using the present analysis it can be shown that the system will not enter a steady state if the maximal velocity of any forward reaction is less than the steady state flux through the system. This condition represents a practical test for determining if a system will enter steady state but is valid only when the rate of the primary enzyme is not affected allosterically be intermediates in the pathway. The equations are used to analyse a portion of the rat liver glycogenic pathway that catalyses the conversion of glucose to fructose 1,6-bisphosphate.

Animals↗
PubMed1984

Evidence for a specific phosphoryl binding site in swine kidney phosphofructokinase.

Phosphofructokinase (PFK) from swine kidney was purified by a procedure which included affinity chromatography on Cibacron blue F3GA-Sepharose 4B and ATP-Sepharose 4B columns in order to examine its binding properties. The homogeneous enzyme was purified more than 3000-fold with a yield of 30% and it had a specific activity of 39.8 mumol/min/mg of protein at 25 degrees C. The molecular weight of the native enzyme was 360 000 and it contained 4 identical subunits of molecular weight 88 000. The principal catalytically reacting form of the enzyme had a S20,w of 13.7 S which corresponds to a molecular weight of 360 000 +/- 6 000. The initial velocity patterns in the forward and reverse directions suggested a sequential mechanism for the reaction. The Km values for fructose 6-phosphate, ATP, fructose, 1,6-bisP and ADP were 33 microM, 8.3 microM, 460 microM and 110 microM, respectively. The homogeneous native enzyme binds specifically to phosphoryl groups immobilized in cellulose phosphate columns. ATP and fructose 6-phosphate interacted with the enzyme and decreased its affinity for phosphoryl binding sites. Other metabolites including fructose 1,6-bisP, glucose 6-phosphate and various nucleotides, alone or in various combinations, were ineffective in promoting the dissociation of the enzyme. Allosteric effectors of the enzyme, such as citrate and AMP were also inactive. However, the cooperatively altered the concentration of ATP required to dissociate the enzyme from phosphoryl groups. The bound enzyme was enzymatically inactive. The enzyme was also inactivated when it was treated with pyridoxal 5'-phosphate and reduced with sodium borohydride and the inactive enzyme no longer bound to cellulose phosphate. These effects were not observed when treatment with pyridoxal 5'-phosphate was carried out in the presence of fructose 6-phosphate. These observations and the results of similar studies with swine kidney fructose 1,6-bisphosphatase (FBPase) show that both enzymes share the unique property of binding specifically to phosphoryl groups. FBPase interacts through its allosteric AMP binding site and PFK binds through its fructose 6-P binding site. This specific binding of both enzymes through these sites result in the inactivation of PFK and the desensitization of FBPase to allosteric inhibition by AMP. In the unbound state PFK may be active and FBPase can be inhibited by AMP. Taken collectively, these binding effects could play a role in the reciprocal regulation of these enzymes during gluconeogenesis in kidney.

Adenosine Triphosphate↗
PubMed1984

Binding and regulatory properties of phosphofructokinase from swine kidney.

The influence of fructose 2,6-bisphosphate on the activation of purified swine kidney phosphofructokinase as a function of the concentration of fructose 6P, ATP and citrate was investigated. The purified enzyme was nearly completely inhibited in the presence of 2 mM ATP. The addition of 20 nM fructose 2,6-P2 reversed the inhibition and restored more than 80% of the activity. In the absence of fructose 2,6-P2 the reaction showed a sigmoidal dependence on fructose-6-phosphate. The addition of 10 nM fructose 2,6-bisphosphate decreased the K0.5 for fructose 6-phosphate from 3 mM to 0.4 mM in the presence of 1.5 mM ATP. These results clearly show that fructose 2,6-bisphosphate increases the affinity of the enzyme for fructose 6-phosphate and decreases the inhibitory effect of ATP. The extent of inhibition by citrate was also significantly decreased in the presence of fructose 2,6-phosphate. The influence of various effectors of phosphofructokinase on the binding of ATP and fructose 6-P to the enzyme was examined in gel filtration studies. It was found that kidney phosphofructokinase binds 5.6 moles of fructose 6-P per mole of enzyme, which corresponds to about one site per subunit of tetrameric enzyme. The KD for fructose 6-P was 13 microM and in the presence of 0.5 mM ATP it increased to 27 microM. The addition of 0.3 mM citrate also increased the KD for fructose 6-P to about 40 microM. AMP, 10 microM, decreased the KD to 5 microM and the addition of fructose 2,6-phosphate decreased the KD for fructose 6-P to 0.9 microM. The addition of these compounds did not effect the maximal amount of fructose 6-P bound to the enzyme, which indicated that the binding site for these compounds might be near, but was not identical to the fructose 6-P binding site. The enzyme bound a maximum of about 12.5 moles of ATP per mole, which corresponds to 3 moles per subunit. The KD of the site with the highest affinity for ATP was 4 microM, and it increased to 15 microM in the presence of fructose 2,6-bisphosphate. The addition of 50 microM fructose 1,6-bisphosphate increased the KD for ATP to 5.9 microM. AMP increased the KD to 5.9 microM whereas 0.3 mM citrate decreased the KD for ATP to about 2 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗
PubMed1983

A review of animal phosphofructokinase isozymes with an emphasis on their physiological role.

Phosphofructokinase (PFK) isozymes and their physiological significance have been the focus of extensive research. The majority of this work has been centered around the PFK isozymes of rat, human and rabbit tissues. Consequently, this review emphasizes these studies. Additionally, a review of PFK isozymes in chickens, mice, guinea pig, and pig is presented. The relationship of the properties of each PFK isozyme in different tissues to the rates of glycolysis and/or gluconeogenesis in those tissues is discussed where possible. Moreover, the contribution of the different PFK isoenzymes to alterations of the glycolytic rate in various tissues is discussed in relationship to variations in nutritional, hormonal, developmental or pathological status of the animal.

Animals↗
PubMed1992

Oscillations in glycolysis: multifactorial quantitative analysis in muscle extract.

A multifactorial quantitative analysis of oscillations in glycolysis was conducted in the postmicrosomal supernatant of rat muscle homogenates incubated in the presence of yeast hexokinase. Oscillations in adenine nucleotides, D-fructose 1,6-bisphosphate, triose phosphates, L-glycerol 3-phosphate, 3HOH generation from D-[5-3H]glucose, NADH and L-lactate production were documented. The occurrence of such oscillations were found to depend mainly on the balance between the consumption of ATP associated with the phosphorylation of D-glucose, as catalyzed by both yeast and muscle hexokinase, and the net production of ATP resulting from the further catabolism of D-fructose 6-phosphate, as initiated by activation of phosphofructokinase. The oscillatory pattern was suppressed in the presence of D-fructose 2,6-bisphosphate. It is proposed that the quantitative information gathered in this study may set the scene for further studies in extracts of cells other than myocytes, e.g. hepatocytes and pancreatic islet cells, in which no oscillation of glycolysis was so far observed.

Adenine Nucleotides↗
PubMed1996

Fructose-1,6-bisphosphate as a metabolic substrate in hog ileum smooth muscle during hypoxia.

Exogenously applied fructose-1,6-bisphosphate has been reported to be effective in preventing some damage to the small intestine during ischemia. To determine whether exogenously applied fructose-1,6-bisphosphate protects ileum smooth muscle from damage from hypoxia and from reoxygenation, we examined the effect of fructose-1,6-bisphosphate on the ability of hog ileum smooth muscle to maintain isometric force during hypoxia and to generate isometric force after reoxygenation in the presence of 5 mM glucose. After 180 min of hypoxia, tissues incubated with 20 mM fructose-1,6-bisphosphate maintained significantly greater levels of isometric force than tissues incubated in the absence of exogenous substrate (23% of pre-hypoxia force compared to 16%). During the first contraction following reoxygenation there was a significantly greater force generation in tissues incubated with 20 mM fructose-1,6-bisphosphate during the hypoxia period compared to tissues with no exogenous substrate included during the hypoxia period (29% of pre-hypoxia force compared to 19%). However, glucose always was a better metabolic substrate compared to fructose-1,6-bisphosphate under all experimental conditions. The presence of fructose-1,6-bisphosphate during hypoxia likely improved tissue function by fructose-1,6-bisphosphate entering the cells and acting as a glycolytic intermediate, since during a 120 min period of hypoxia, unmounted ileum smooth muscle metabolized 1,6-13C-fructose-1,6-bisphosphate to 3-13C-lactate. This conversion of 1,6-13C-fructose-1,6-bisphosphate to 3-13C-lactate was inhibited by the addition of 1 mM iodoacetic acid, a glycolytic inhibitor. We conclude that exogenously provided fructose-1,6-bisphosphate does provide modest protection of ileum smooth muscle from hypoxic damage by functioning as a glycolytic intermediate and improving the cellular energy state.

Animals↗
PubMed1993

Metabolic effects of benzoate and sorbate in the yeast Saccharomyces cerevisiae at neutral pH.

Preincubation of yeast cells in the presence of benzoate or sorbate at an extracellular pH value of 6.8 elicited a set of metabolic effects on sugar metabolism, which became apparent after the subsequent glucose addition. They can be summarized as follows: a) reduced glucose consumption; b) inhibition of glucose- and fructose-phosphorylating activities; c) suppression of glucose-triggered peak of hexoses monophosphates; d) substantial reduction of glucose-triggered peak of fructose 2,6-bisphosphate; e) block of catabolite inactivation of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxy-kinase, but not of cytoplasmic malate dehydrogenase. On the whole this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenetic to a glycolytic state. Our data also show that, unlike former assumptions, intracellular acidification is not likely to mediate the bulk of metabolic effects of benzoate and sorbate, since under our working conditions intracellular pH kept close to neutrality.

Benzoates↗
PubMed1993

6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification, kinetics and immunological properties.

Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55,800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53,900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53,300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mumol.l-1).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Effect of work-induced hypertrophy on muscle glucose metabolism in lean and obese mice.

The effect of work-induced hypertrophy (without any concomitant change in circulating parameters) on skeletal muscle metabolism was studied in lean mice and in gold-thioglucose-obese mice. Soleus muscle was functionally overloaded in one leg by tenotomy of gastrocnemius muscle 4 days before muscle isolation, muscle in the other leg being used as control. Basal deoxyglucose uptake and glycolysis were markedly increased in overloaded muscles compared with control muscles, together with a ten-fold increase in fructose 2-6 bisphosphate content. In the presence of maximally effective insulin concentrations, deoxyglucose uptake and glycolysis were identical in overloaded and control muscles of lean mice, while the effects of overload and insulin were partly additive in muscles of gold-thioglucose-obese mice. The sensitivity to insulin and insulin binding to muscles were not modified in overloaded muscles. Insulin-stimulated glycogenogenesis was decreased by about 50% probably due to a lower amount of glycogen synthase in overloaded than in control muscles. Thus, in muscles of gold-thioglucose-obese mice work-induced hypertrophy increased the response to maximal insulin concentrations without modifying the altered insulin sensitivity and decreased insulin binding.

Animals↗
PubMed1977

Fructose-1,6-bisphosphate, a regulator of metabolism.

Fructose-1,6-bisphosphate affects the rate of a large variety of enzyme reactions. In some instances its role as a physiologic effector is well documented. In many cases the effects of fructose bishosphate on particular enzymes have been demonstrated in vitro but the link to physiologic conditions has not yet been established. It is the purpose of this paper to summarize the scattered findings in fructose bisphosphate as an effector of enzyme reactions and to draw some conclusions about the role of the compound in metabolic regulation.

Carbohydrate Metabolism↗
PubMed1993

Studies on the expression of the TNF alpha receptors (p55 and p75) and their relative contributions to prostanoid production and glycolytic rate by rheumatoid synovial fibroblasts in vitro.

The effect of monoclonal antibodies against the 55- and 75-kDa (p55 and p75) tumour necrosis factor (TNF) receptors on two tumour necrosis factor alpha (TNF alpha) induced responses was studied in rheumatoid synovial fibroblasts (RSF) in vitro. This provided functional evidence for the expression of both receptor types, which was confirmed by substantial inhibition of 125I-TNF alpha binding to RSF by both antibodies. TNF alpha stimulation of prostanoid production was found to be a function of ligand binding to both receptor types, whereas the stimulation of cellular glycolysis was largely mediated via the p55 receptor. Thus, we showed that RSF express both types of TNF receptor and functional studies showed that stimulation of each receptor results in both similar and dissimilar cellular responses.

Antibodies, Monoclonal↗