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Biomedical subjects

L Hue

Publications and source records attributed to L Hue.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis.

Adenosine Triphosphate↗
PubMed1997

Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase.

Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted.

Adenylate Kinase↗
PubMed1997

Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

In a structural model of the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase based on the analogy with adenylate kinase, Lys-174, Asp-179 and Asp-191 residues are located in the putative active site. Asp-179 and Asp-191 are conserved in all known 6-phosphofructo-2-kinase sequences. In contrast, Lys-174 is conserved except in a yeast isoenzyme, fbp26, where it is replaced by glycine. Yeast fbp26 possesses fructose-2,6-bisphosphatase activity, but is devoid of 6-phosphofructo-2-kinase activity. Mutation of Asp-179 and Asp-191 of the rat liver isoenzyme to alanine increased the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate 2000- and 1000-fold respectively, whereas mutation of Lys-174 to glycine decreased the Vmax of 6-phosphofructo-2-kinase more than 4000-fold. In contrast, none of the mutations affected the kinetic parameters of fructose-2,6-bisphosphatase. CD and fluorescence measurements indicated that the mutations had no effect on the structure and stability of the recombinant proteins. The results show that Asp-179 and Asp-191 participate in fructose 6-phosphate binding, whereas Lys-174 is important for catalysis. Therefore the natural mutation of Lys-174 to glycine in the fbp26 yeast isoenzyme could explain the lack of 6-phosphofructo-2-kinase activity. These results support a novel 6-phosphofructo-2-kinase structure model based on adenylate kinase.

Adenosine Triphosphate↗
PubMed1997

Inhibition of glycogenolysis by a glucose analogue in the working rat heart.

The effects of BAY o 1248, an inhibitor of alpha-amylo-1, 6-glucosidase, on glycogenolysis and post-ischemic functional recovery were investigated in isolated perfused rat hearts. Working rat hearts were perfused during 30 min with 11 mm glucose (controls) and, in some hearts, with 1 microM insulin or 5 mM lactate to increase their glycogen concentration. The hearts were then submitted to 10 min of no-flow ischemia and reperfused during 15 min with 11 mM glucose alone. Glycogen content was increased by 50% in hearts perfused with insulin or lactate. During ischemia, glycogen breakdown was similar in the control and lactate groups, but was abolished in the insulin-group. At reperfusion, functional recovery was improved in glycogen-loaded hearts compared to controls. When hearts were perfused with 1 mM BAY o 1248, added before ischemia, glycogenolysis was inhibited in the three groups and functional recovery was hampered in both the control and lactate groups. In the insulin group, however, the functional recovery was barely affected by BAY o 1248. We conclude that: (i) BAY o 1248 is an inhibitor of heart glycogen breakdown; (ii) the consequences of inhibition of ischemic glycogenolysis on post-ischemic functional recovery depend on the conditions; and (iii) the protective effect of insulin does not result from ischemic glycogenolysis.

1-Deoxynojirimycin↗
PubMed1997

Activation of nitric oxide synthase by ischaemia in the perfused heart.

OBJECTIVE: Recent data have indicated that the activity of nitric oxide synthase (NO synthase), the enzyme producing NO from L-arginine, could be modified by ischaemia. The aim of the present work was therefore to study whether ischaemia activated NO synthase. METHODS: NO synthase activity was measured by the conversion of radioactive arginine into citrulline in extracts of isolated perfused rabbit hearts submitted to low-flow ischaemia and reperfusion. RESULTS: When measured in heart homogenates, NO synthase activity was significantly increased during ischaemia. This activation was already detectable after 5 min of ischaemia and was maintained during the whole ischaemic period. After cell fractionation, NO synthase was recovered in cytosolic and membrane fractions. The increase in NO synthase activity by ischaemia was related to an activation of the cytosolic activity, while the membrane-bound NO synthase activity remained constant. CONCLUSION: NO synthase activity in the heart is rapidly stimulated by ischaemia and this stimulation is maintained during the whole ischaemic episode. This activation is found only in the cytosolic fraction, whereas the particulate activity is not affected by ischaemia.

Animals↗
PubMed1996

Signaling pathway involved in the activation of heart 6-phosphofructo-2-kinase by insulin.

Incubation of isolated rat cardiomyocytes with insulin increased 2-deoxyglucose uptake, glycogen synthesis, and fructose 2, 6-bisphosphate content. Half-maximal effects were obtained with 1-2 nM insulin. The insulin-induced increase in fructose 2,6-bisphosphate content was preceded by a 2-3-fold activation of 6-phosphofructo-2-kinase, which was independent of glucose transport. Insulin activated phosphatidylinositol 3-kinase and p70 ribosomal S6 kinase (p70 S6 kinase), but had no significant effect on mitogen-activated protein kinase, although phorbol 12-myristate 13-acetate activated the latter. The effect of insulin on fructose 2, 6-bisphosphate, 6-phosphofructo-2-kinase, and phosphatidylinositol 3-kinase was blocked by wortmannin. However, rapamycin, which inhibited p70 S6 kinase activation, and PD 98059, an inhibitor of the mitogen-activated protein kinase pathway, had no effect on the insulin-induced activation of 6-phosphofructo-2-kinase. Heart 6-phosphofructo-2-kinase can therefore be regarded as a glycolytic target of insulin. Its activation by insulin might be mediated by phosphatidylinositol 3-kinase.

Androstadienes↗
PubMed1996

The ATP-binding site in the 2-kinase domain of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the role of Lys-54 and Thr-55 by site-directed mutagenesis.

All known 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes contain a sequence (GX4GK(S/T)) in the 6-phosphofructo-2-kinase domain corresponding to the so-called nucleotide binding fold signature or Walker A motif. Mutagenesis and crystal structure data from several nucleotide binding proteins, which also contain this sequence, showed the importance of the lysine and serine/threonine residues in nucleotide binding. We have studied the role of Lys-54 and Thr-55 in MgATP binding in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis. Lys-54 was mutated to methionine, whereas Thr-55 was mutated to valine, serine, and cysteine. Three mutants, Lys-54 to Met and Thr-55 to Cys or Val, displayed more than a 5000-fold decrease in 6-phosphofructo-2-kinase activity compared with the wild type. The mutations had no effect on fructose-2, 6-bisphosphatase activity and did not affect the activation of fructose-2,6-bisphosphatase after phosphorylation by cyclic 3', 5'-AMP-dependent protein kinase. Binding experiments with ATP, ADP, and their analogs (3'-N-methylanthraniloyl derivatives) showed that these two residues do not play the same role. Lys-54 is involved in ATP binding, whereas Thr-55 is important for catalysis.

Adenosine Diphosphate↗
PubMed1996

Protein kinase signaling pathway triggered by cell swelling and involved in the activation of glycogen synthase and acetyl-CoA carboxylase in isolated rat hepatocytes.

Incubation of isolated hepatocytes with glutamine or proline or in hypotonic media is known to activate glycogen synthase and acetyl-CoA carboxylase as a result of cell swelling. We report here that the same experimental conditions caused an activation of phosphatidylinositol 3-kinase and p70 ribosomal protein S6 kinase (p70 S6 kinase) but did not modify the activity of p42 mitogen-activated protein kinase. In addition, rapamycin, an inhibitor of p70 S6 kinase activation, prevented the amino acid- and hypotonicity-induced activation of p70 S6 kinase but did not block the activation of glycogen synthase and acetyl-CoA carboxylase, thus ruling out p70 S6 kinase as a necessary component in the activation pathway. By contrast, wortmannin or LY294002, inhibitors of phosphatidylinositol 3-kinase, completely blocked the activation of phosphatidylinositol 3-kinase and p70 S6 kinase and partly blocked the activation of glycogen synthase and acetyl-CoA carboxylase. Therefore, phosphatidylinositol 3-kinase might be a component of the signaling pathway that is triggered by cell swelling and is responsible, at least in part, for the activation of glycogen synthase and acetyl-CoA carboxylase. Incubation of hepatocytes with 0.1 microM epidermal growth factor doubled the activity of p42 mitogen-activated protein kinase without activating glycogen synthase.

Acetyl-CoA Carboxylase↗
PubMed1996

Activation of hepatic acetyl-CoA carboxylase by glutamate and Mg2+ is mediated by protein phosphatase-2A.

The activation of hepatic acetyl-CoA carboxylase by Na(+)-cotransported amino acids such as glutamine has been attributed mainly to the stimulation of its dephosphorylation by accumulating dicarboxylic acids, e.g. glutamate. We report here on a hepatic species of protein phosphatase-2A that activates acetyl-CoA carboxylase in the presence of physiological concentrations of glutamate or Mg2+ and, under these conditions, accounts for virtually all the hepatic acetyl-CoA carboxylase phosphatase activity. Glutamate also stimulated the dephosphorylation of a synthetic pentadecapeptide encompassing the Ser-79 phosphorylation site of rat acetyl-CoA carboxylase, but did not affect the dephosphorylation of other substrates such as phosphorylase. Conversely, protamine, which stimulated the dephosphorylation of phosphorylase, inhibited the activation of acetyl-CoA carboxylase. A comparison with various species of muscle protein phosphatase-2A showed that the stimulatory effects of glutamate and Mg2+ on the acetyl-CoA carboxylase phosphatase activity are largely mediated by the regulatory A subunit. Glutamate and Mg2+ emerge from our study as novel regulators of protein phosphatase-2A when acting on acetyl-CoA carboxylase.

AMP-Activated Protein Kinases↗
PubMed1995

The antianginal agent ranolazine is a weak inhibitor of the respiratory complex I, but with greater potency in broken or uncoupled than in coupled mitochondria.

Ranolazine (RS-43285) has shown antianginal effects in clinical trials and cardiac anti-ischaemic activity in several in vivo and in vitro animal models, but without affecting haemodynamics. Its mechanism is thought to mainly involve a switch in substrate utilisation from fatty acids to glucose to, thus, improve efficiency of O2 use; however, its precise molecular target(s) are unknown. In studies to investigate its action further, using isolated rat heart mitochondria, ranolazine was found to weakly inhibit (pIC50 values > 300 microM) respiration by coupled mitochondria provided with NAD(+)-linked substrates but not with succinate. With broken mitochondrial membranes or submitochondrial particles, ranolazine inhibited NADH but not succinate oxidation and with pIC50 values in the lower range of 3-50 microM. Studies with different electron acceptors and respiratory inhibitors indicated that it inhibits respiratory Complex I at a site between ferricyanide and menadione and ubiquinone-1 reduction (i.e. at a similar locus to rotenone). However, unlike rotenone, ranolazine was an uncompetitive inhibitor with respect to ubiquinone-1. Ranolazine inhibition of Complex I was reversible and occurred also with mitochondria from pig, guinea pig, and human heart, and rat liver. Further studies using rat heart mitochondria in different energisation states (i.e. broken, uncoupled, or coupled) showed a 50-100-fold shift to greater potency of ranolazine in the broken compared to the coupled; with the uncoupled it was about 2-fold less potent than the broken. These shifts in potency were not found with rotenone or amytal. Studies with radiolabelled ranolazine showed that it bound to mitochondrial membranes with greater affinity in the broken compared to the coupled or uncoupled conditions. Rotenone displaced radiolabelled ranolazine from its binding site. This property of ranolazine may play some role in its anti-ischaemic activity.

Acetanilides↗
PubMed1995

Protection against ischemic injury by nonvasoactive concentrations of nitric oxide synthase inhibitors in the perfused rabbit heart.

BACKGROUND: The functional and metabolic effects of inhibitors of nitric oxide (NO) synthase on ischemic hearts have not been investigated. This work was designed to perform such a study in isolated perfused rabbit hearts submitted to low-flow ischemia. METHODS AND RESULTS: After a 30-minute equilibration period, the hearts were submitted to low-flow ischemia for 60 minutes followed by reperfusion for 30 minutes. Functional and metabolic parameters were followed in hearts perfused with or without inhibitors of NO synthase or NO precursors, which were added 15 minutes before ischemia but were absent during reperfusion. Ischemic contracture was delayed and reduced in hearts perfused with 1 mumol/L L-N-monomethylarginine (L-NMMA) or 1 mumol/L L-N-arginine methylester, two inhibitors of NO synthase, but not with D-N-monomethylarginine, the inactive enantiomer of L-NMMA. The protection was suppressed by addition to the perfusate containing L-NMMA of 1 mmol/L L-arginine or 0.1 mmol/L sodium nitroprusside but not by addition of 10 mumol/L 8-bromo cGMP, a cGMP analogue. The functional protection by 1 mumol/L L-NMMA was related to a stimulation of glycolysis from exogenous glucose and a preservation of the glycogen stores. This resulted in a better maintenance of high-energy phosphates and a lower acidosis as measured by 31P nuclear magnetic resonance spectroscopy. During reperfusion, functional recovery was more than doubled, and enzyme release was halved in L-NMMA-treated hearts compared with controls. The functional and metabolic protection was maximal at 1 nmol/L to 1 mumol/L L-NMMA, ie, below the vasoactive concentrations of the inhibitor. CONCLUSIONS: Nonvasoactive concentrations of NO synthase inhibitors protect the heart against ischemic damage; this relates to a stimulation of glycolysis from exogenous glucose.

Adenosine Triphosphate↗
PubMed1995

Trimetazidine effects on the damage to mitochondrial functions caused by ischemia and reperfusion.

Trimetazidine (TMZ) is an anti-ischemic compound whose precise mode of action is unknown, although several studies have suggested a metabolic effect, and there have been reports of protection of mitochondria against oxidative stress damage. Using a Langendorff rat heart model, we examined the effects of TMZ on the mitochondrial damage following 30 minutes of ischemia and 5 minutes of reperfusion. Mitochondrial respiration with succinate, glutamate-malate and ascorbate-N,N,N',N'-tetramethylphenylenediamine (TMPD) as substrates was significantly decreased following ischemia-reperfusion. Preperfusion with 10(-5) M TMZ had no effect on these rates in normoxic or ischemic hearts. However, 10(-3) M TMZ significantly decreased the glutamate-malate rate in mitochondria from normoxic hearts, and this rate was not further decreased following ischemia-reperfusion, and 10(-3) M TMZ also partially protected ascorbate-TMPD activity. The effect on glutamate-malate was probably due to an inhibition of complex I by TMZ, which specifically inhibited reduced nicotinamide-adenine-dinucleotide-cytochrome c reductase and complex I in lysed mitochondria. We also studied the effects of TMZ on the activity of pyruvate dehydrogenase (PDH) in normoxic and ischemic hearts perfused with 0.5 mM palmitate, which caused the enzyme to be almost completely inactivated. After short periods of ischemia (10-20 minutes) the PDH inactivation by palmitate was progressively lost. Preperfusion with 10(-5) M TMZ had a tendency to decrease lactate dehydrogenase release, accompanied by a maintenance of the inhibition of PDH by palmitate. This may allow the heart to oxidize fatty acids preferentially during reperfusion, hence removing possible toxic acyl esters.

Animals↗
PubMed1995

Study of the roles of Arg-104 and Arg-225 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis.

The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) have been studied by site-directed mutagenesis. In recombinant rat liver PFK-2/FBPase-2, mutation of Arg-225 to Ser increased the Km of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentrations between pH 6 and 9.5. The mutation had no effect on the Vmax of PFK-2 or on the Km of PFK-2 for MgATP. The mutation also increased the Vmax. of FBPase-2 4-fold without changing the Km for Fru-2,6-P2 or IC50 of Fru-6-P. These findings are in agreement with a previous study [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protection by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and suggest that Arg-225 participates in Fru-6-P binding. In recombinant rat muscle PFK-2/FBPase-2, mutation of Arg-104 to Ser increased the Km for Fru-6-P 60-fold, increased the IC50 of citrate, increased the Vmax. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the Km of PFK-2 for MgATP. The mutation also decreased the Vmax. of FBPase-2 3-fold, increased the Km for Fru-2,6-P2 70-fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity was more sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is involved in Fru-6-P binding in the PFK-2 domain and that it might also bind citrate. Structural changes resulting from the mutation might be responsible for the changes in kinetic properties of FBPase-2.

Animals↗
PubMed1995

[Regulation of liver carbohydrate and lipid metabolism by cell volume].

Certain amino acids such as glutamine and proline stimulate liver glycogen synthesis and lipogenesis. This implies cell swelling and leads to the activation of glycogen synthase and acetyl-CoA carboxylase by a common mechanism. This mechanism results from the cell response to swelling and involves a fall in intracellular concentration of chloride ions and an increase in intracellular concentration of glutamate and aspartate. These ions indeed regulate the protein phosphatases that activate glycogen synthase and acetyl-CoA carboxylase.

Acetyl-CoA Carboxylase↗
PubMed1994

Cyclic GMP in the perfused rat heart. Effect of ischaemia, anoxia and nitric oxide synthase inhibitor.

Working rat hearts perfused with 5.5 mM glucose were submitted to a 10-min period of no-flow ischaemia or anoxia. Both conditions stimulated glycogenolysis, activated phosphorylase and increased cyclic GMP content, although the time course of these changes differed in anoxia and ischaemia. Changes in cyclic GMP content were not correlated with glycogenolysis or phosphorylase activation. Perfusion with 1 microM L-nitroarginine methylester, an inhibitor of nitric oxide synthase, decreased cGMP concentration under normoxic conditions and abolished the ischaemia-induced increase in cGMP. The inhibitor decreased the coronary flow without affecting the overall working performance of the hearts under normoxic conditions.

Adenosine Triphosphate↗
PubMed1994

Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain.

Asp-130 of the recombinant skeletal-muscle 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase was mutated into Ala in order to study its role in catalysis and/or substrate binding. The D130A mutant displayed a 30- to 140-fold decreased 2-kinase Vmax, depending on the pH, and a 30- and 60-fold increase in Km for MgATP and Fru-6-P respectively at pH 8.5 compared with the wild-type. Mutagenesis of Asp-130 to Ala had no effect on the 2-phosphatase activity, and fluorescence measurements indicated that the changes in kinetic properties of PFK-2 in the D130A mutant were not due to instability. The role of Asp-130 in the 2-kinase reaction is discussed and compared with that of Asp-103 of 6-phosphofructo-1-kinase from Escherichia coli, which binds Mg2+.

Amino Acid Sequence↗