The Experts below are selected from a list of 99 Experts worldwide ranked by ideXlab platform
E Van Schaftingen - One of the best experts on this subject based on the ideXlab platform.
-
Investigation on the mechanism by which Fructose, hexitols and other compounds regulate the translocation of glucokinase in rat hepatocytes
Biochemical Journal, 1997Co-Authors: L. Niculescu, Maria Veiga-da-cunha, E Van SchaftingenAbstract:In isolated hepatocytes in suspension, the effect of sorbitol but not that of Fructose to increase the concentration of Fructose 1-Phosphate and to stimulate glucokinase was abolished by 2-hydroxymethyl-4-(4-N,N-dimethylamino-1-piperazino)-pyrimidine (SDI 158), an inhibitor of sorbitol dehydrogenase. In hepatocytes in primary culture, Fructose was metabolized at approximately one-quarter of the rate of sorbitol, and was therefore much less potent than the polyol in increasing the concentration of Fructose 1-Phosphate and the translocation of glucokinase. In cultures, sorbitol, commercial mannitol, Fructose, D-glyceraldehyde or high concentrations of glucose caused Fructose 1-Phosphate formation and glucokinase translocation in parallel. Commercial mannitol was contaminated by approx. 1% sorbitol, which accounted for its effects. The effects of sorbitol, Fructose and elevated concentrations of glucose were partly inhibited by ethanol, glycerol and glucosamine. Mannoheptulose increased translocation without affecting Fructose 1-Phosphate concentration. Kinetic studies performed with recombinant human beta-cell glucokinase indicated that this sugar, in contrast with N-acetylglucosamine, binds to glucokinase competitively with the regulatory protein. All these observations indicate that translocation is promoted by agents that favour the dissociation of the glucokinase-regulatory-protein complex either by binding to the regulatory protein (Fructose I-Phosphate) or to glucokinase (glucose, mannoheptulose). They support the hypothesis that the regulatory protein of glucokinase acts as an anchor for this enzyme that slows down its release from digitonin-permeabilized cells.
-
binding of sorbitol 6 Phosphate and of Fructose 1 Phosphate to the regulatory protein of liver glucokinase
Biochemical Journal, 1992Co-Authors: Annick Vandercammen, Michel Detheux, E Van SchaftingenAbstract:Using a binding assay in which the ligand-protein complex is separated from free ligand by precipitation with poly(ethylene glycol) 6000, we found that the regulatory protein of rat liver glucokinase bound close to 1 mol of radiolabelled sorbitol 6-Phosphate, a negative effector, or of Fructose 1-Phosphate, a positive effector, per mol of regulatory protein. Scatchard plots were linear, the dissociation constant being 0.3 microM for both Phosphate esters. Sorbitol 6-Phosphate and Fructose 1-Phosphate competed with each other for the binding. Competition was also observed with psicose 1-Phosphate, ribitol 5-Phosphate, arabitol 5-Phosphate and 3-phosphoglycerate, all of which are known to affect the inhibition exerted by the regulatory protein. At a concentration of 10%, poly(ethylene glycol) 6000 decreased the concentration of regulatory protein causing 50% inhibition to a larger extent in the absence (12-fold) than in the presence (3-fold) of a saturating concentration of Fructose 6-Phosphate, another negative effector. Furthermore, it increased by about 3-fold the apparent affinity for inhibitory Phosphate esters, indicating that it induced conformational changes of the regulatory protein.
-
The regulatory protein of liver glucokinase
Advances in Enzyme Regulation, 1992Co-Authors: E Van Schaftingen, Annick Vandercammen, Michel Detheux, Dewi R. DaviesAbstract:Fructose, sorbitol and D-glyceraldehyde stimulate the rate of glucose phosphorylation in isolated hepatocytes. This effect is mediated by Fructose 1-Phosphate, which releases the inhibition exerted by a regulatory protein on liver glucokinase. In the presence of Fructose 6-Phosphate, the regulatory protein binds to, and inhibits, liver glucokinase. Fructose 1-Phosphate antagonizes this inhibition by causing dissociation of the glucokinase-regulatory protein complex. Both Phosphate esters act by binding to the regulatory protein, and by presumably causing changes in its conformation. The regulatory protein behaves as a fully competitive inhibitor. It inhibits liver glucokinase from various species, and rat islet glucokinase, but has no effect on hexokinases from mammalian tissues or from yeast, or on glucokinase from microorganisms. Kinetic studies indicate that the regulatory protein binds to glucokinase at a site distinct from the catalytic site. Several Phosphate esters, mainly polyol-Phosphates, were found to mimick the effect of Fructose 6-Phosphate. The most potent is sorbitol 6-Phosphate, suggesting that Fructose 6-Phosphate is recognized by the regulatory protein in its open-chain configuration. Other Phosphate esters and Pi have a Fructose 1-Phosphate-like effect. The stimulatory effect of Fructose on glucose phosphorylation is observed not only in isolated hepatocytes but also in the livers of anesthetized rats. This suggests that Fructose could be a nutritional signal causing an increase in the hepatic glucose uptake.
Milton H. Saier - One of the best experts on this subject based on the ideXlab platform.
-
Identification of a phosphoenolpyruvate:Fructose phosphotransferase system (Fructose-1-Phosphate forming) in Listeria monocytogenes.
Journal of bacteriology, 1993Co-Authors: W J Mitchell, Jonathan Reizer, Christopher D. Herring, C Hoischen, Milton H. SaierAbstract:Listeria monocytogenes is a gram-positive bacterium whose carbohydrate metabolic pathways are poorly understood. We provide evidence for an inducible phosphoenolpyruvate (PEP):Fructose phosphotransferase system (PTS) in this pathogen. The system consists of enzyme I, HPr, and a Fructose-specific enzyme II complex which generates Fructose-1-Phosphate as the cytoplasmic product of the PTS-catalyzed vectorial phosphorylation reaction. Fructose-1-Phosphate kinase then converts the product of the PTS reaction to Fructose-1,6-bisPhosphate. HPr was shown to be phosphorylated by [32P]PEP and enzyme I as well as by [32P]ATP and a Fructose-1,6-bisPhosphate-activated HPr kinase like those found in other gram-positive bacteria. Enzyme I, HPr, and the enzyme II complex of the Listeria PTS exhibit enzymatic cross-reactivity with PTS enzyme constituents from Bacillus subtilis and Staphylococcus aureus.
-
nucleotide sequence of the rhodobacter capsulatus fruk gene which encodes Fructose 1 Phosphate kinase evidence for a kinase superfamily including both phosphofructokinases of escherichia coli
Journal of Bacteriology, 1991Co-Authors: Longfei Wu, Jonathan Reizer, Aiala Reizer, John M Tomich, Milton H. SaierAbstract:Abstract The fruK gene encoding Fructose-1-Phosphate kinase (FruK), located within the Fructose (fru)-catabolic operon of Rhodobacter capsulatus, was sequenced. FruK of R. capsulatus (316 amino acids; molecular weight = 31,232) is the same size as and is homologous to FruK of Escherichia coli, phosphofructokinase B (PfkB) of E. coli, phosphotagatokinase of Staphylococcus aureus, and ribokinase of E. coli. These proteins therefore make up a family of homologous proteins, termed the PfkB family. A phylogenetic tree for this new family was constructed. Sequence comparisons plus chemical inactivation studies suggested the lack of involvement of specific residues in catalysis. Although the Rhodobacter FruK differed markedly from the other enzymes within the PfkB family with respect to amino acid composition, these enzymes exhibited similar predicted secondary structural features. A large internal segment of the Rhodobacter FruK was found to be similar in sequence to the domain bearing the sugar bisPhosphate-binding region of the large subunit of ribulose 1,5-bisPhosphate carboxylase/oxygenase of plants and bacteria. Proteins of the PfkB family did not exhibit statistically significant sequence identity with PfkA of E. coli. PfkA, however, is homologous to other prokaryotic and eukaryotic ATP- and PPi-dependent Pfks (the PfkA family). These eukaryotic, ATP-dependent enzymes each consist of a homotetramer (mammalian) or a heterooctamer (yeasts), with each subunit containing an internal duplication of the size of the entire PfkA protein of E. coli. In some of these enzymes, additional domains are present. A phylogenetic tree was constructed for the PfkA family and revealed that the bacterial enzymes closely resemble the N-terminal domains of the eukaryotic enzyme subunits whereas the C-terminal domains have diverged more extensively. The PPi-dependent Pfk of potato is only distantly related to the ATP-dependent enzymes. On the basis of their similar functions, sizes, predicted secondary structures, and sequences, we suggest that the PfkA and PfkB families share a common evolutionary origin.
Emile Van Schaftingen - One of the best experts on this subject based on the ideXlab platform.
-
Overexpression and purification of Fructose-1-Phosphate kinase from Escherichia coli: application to the assay of Fructose 1-Phosphate.
Protein expression and purification, 2000Co-Authors: Maria Veiga Da Cunha, A. Hoyoux, Emile Van SchaftingenAbstract:Fructose 1-Phosphate is a metabolite that plays a regulatory role in liver and is best measured using an assay based on its conversion to Fructose 1,6-bisPhosphate by a bacterial Fructose-1-Phosphate kinase (Fru1PK). The open reading frame encoding Escherichia coli Fru1PK has been introduced in an expression plasmid (pET3a) based on the T7 promoter-driven system, which was used to overexpress the enzyme. The conditions for the production of soluble Fru1PK were optimized. The purification procedure used involved ammonium sulfate precipitation and chromatography on DEAE-Sepharose and was aimed mostly at stabilizing the enzyme and at freeing Fru1PK from bacterial contaminants that could interfere in the Fructose 1-Phosphate assay. From a 1-liter culture, more than 50 mg protein is obtained. This preparation can be used in an enzymatic assay that measures specifically Fructose 1-Phosphate in tissue extracts.
-
Inhibition of phosphomannose isomerase by Fructose 1-Phosphate: an explanation for defective N-glycosylation in hereditary Fructose intolerance.
Pediatric research, 1996Co-Authors: Jaak Jaeken, Maciej Adamowicz, Michel Pirard, Ewa Pronicka, Emile Van SchaftingenAbstract:Isoelectrofocusing of serum sialotransferrins from patients with untreated hereditary Fructose intolerance (HFI) shows a cathodal shift similar to that in carbohydrate-deficient glycoprotein (CDG) syndrome type I and in untreated galactosemia. This report is on serum lysosomal enzyme abnormalities in untreated HFI that are identical to those found in CDG syndrome type I but different from those in untreated galactosemia. CDG syndrome type I is due to phosphomannomutase deficiency, a defect in the early glycosylation pathway. It was found that Fructose 1-Phosphate is a potent competitive inhibitor (Ki congruent to 40 microM) of phosphomannose isomerase (EC 5.3.1.8), the first enzyme of the N-glycosylation pathway thus explaining the N-glycosylation disturbances in HFI.
-
Cloning and Expression of a Xenopus Liver cDNA Encoding a Fructose‐Phosphate‐Insensitive Regulatory Protein of Glucokinase
European journal of biochemistry, 1994Co-Authors: Maria Veiga-da-cunha, Michel Detheux, Nathalie Watelet, Emile Van SchaftingenAbstract:Xenopus liver contains a protein inhibitor of glucokinase that, in contrast to the mammalian regulatory protein of glucokinase, is insensitive to Fructose 6-Phosphate and Fructose 1-Phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551-556]. The purpose of this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver regulatory protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619-amino-acid protein with a molecular mass of 68695 Da and 57% identity with the rat liver regulatory protein. This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant regulatory protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of glucokinase as the protein purified from Xenopus liver. In contrast to the rat liver regulatory protein, both recombinant and native Xenopus regulatory proteins were insensitive to Fructose 6-Phosphate, Fructose 1-Phosphate and to physiological concentrations of Pi, and they inhibited Xenopus glucokinase with greater affinity than rat glucokinase. These results allow one to conclude that the Fructose-Phosphate-insensitive protein of lower vertebrates is homologous to the Fructose-6-Phosphate-sensitive and Fructose-1-Phosphate-sensitive protein found in mammals.
Michel Detheux - One of the best experts on this subject based on the ideXlab platform.
-
Cloning and Expression of a Xenopus Liver cDNA Encoding a Fructose‐Phosphate‐Insensitive Regulatory Protein of Glucokinase
European journal of biochemistry, 1994Co-Authors: Maria Veiga-da-cunha, Michel Detheux, Nathalie Watelet, Emile Van SchaftingenAbstract:Xenopus liver contains a protein inhibitor of glucokinase that, in contrast to the mammalian regulatory protein of glucokinase, is insensitive to Fructose 6-Phosphate and Fructose 1-Phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551-556]. The purpose of this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver regulatory protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619-amino-acid protein with a molecular mass of 68695 Da and 57% identity with the rat liver regulatory protein. This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant regulatory protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of glucokinase as the protein purified from Xenopus liver. In contrast to the rat liver regulatory protein, both recombinant and native Xenopus regulatory proteins were insensitive to Fructose 6-Phosphate, Fructose 1-Phosphate and to physiological concentrations of Pi, and they inhibited Xenopus glucokinase with greater affinity than rat glucokinase. These results allow one to conclude that the Fructose-Phosphate-insensitive protein of lower vertebrates is homologous to the Fructose-6-Phosphate-sensitive and Fructose-1-Phosphate-sensitive protein found in mammals.
-
binding of sorbitol 6 Phosphate and of Fructose 1 Phosphate to the regulatory protein of liver glucokinase
Biochemical Journal, 1992Co-Authors: Annick Vandercammen, Michel Detheux, E Van SchaftingenAbstract:Using a binding assay in which the ligand-protein complex is separated from free ligand by precipitation with poly(ethylene glycol) 6000, we found that the regulatory protein of rat liver glucokinase bound close to 1 mol of radiolabelled sorbitol 6-Phosphate, a negative effector, or of Fructose 1-Phosphate, a positive effector, per mol of regulatory protein. Scatchard plots were linear, the dissociation constant being 0.3 microM for both Phosphate esters. Sorbitol 6-Phosphate and Fructose 1-Phosphate competed with each other for the binding. Competition was also observed with psicose 1-Phosphate, ribitol 5-Phosphate, arabitol 5-Phosphate and 3-phosphoglycerate, all of which are known to affect the inhibition exerted by the regulatory protein. At a concentration of 10%, poly(ethylene glycol) 6000 decreased the concentration of regulatory protein causing 50% inhibition to a larger extent in the absence (12-fold) than in the presence (3-fold) of a saturating concentration of Fructose 6-Phosphate, another negative effector. Furthermore, it increased by about 3-fold the apparent affinity for inhibitory Phosphate esters, indicating that it induced conformational changes of the regulatory protein.
-
The regulatory protein of liver glucokinase
Advances in Enzyme Regulation, 1992Co-Authors: E Van Schaftingen, Annick Vandercammen, Michel Detheux, Dewi R. DaviesAbstract:Fructose, sorbitol and D-glyceraldehyde stimulate the rate of glucose phosphorylation in isolated hepatocytes. This effect is mediated by Fructose 1-Phosphate, which releases the inhibition exerted by a regulatory protein on liver glucokinase. In the presence of Fructose 6-Phosphate, the regulatory protein binds to, and inhibits, liver glucokinase. Fructose 1-Phosphate antagonizes this inhibition by causing dissociation of the glucokinase-regulatory protein complex. Both Phosphate esters act by binding to the regulatory protein, and by presumably causing changes in its conformation. The regulatory protein behaves as a fully competitive inhibitor. It inhibits liver glucokinase from various species, and rat islet glucokinase, but has no effect on hexokinases from mammalian tissues or from yeast, or on glucokinase from microorganisms. Kinetic studies indicate that the regulatory protein binds to glucokinase at a site distinct from the catalytic site. Several Phosphate esters, mainly polyol-Phosphates, were found to mimick the effect of Fructose 6-Phosphate. The most potent is sorbitol 6-Phosphate, suggesting that Fructose 6-Phosphate is recognized by the regulatory protein in its open-chain configuration. Other Phosphate esters and Pi have a Fructose 1-Phosphate-like effect. The stimulatory effect of Fructose on glucose phosphorylation is observed not only in isolated hepatocytes but also in the livers of anesthetized rats. This suggests that Fructose could be a nutritional signal causing an increase in the hepatic glucose uptake.
Annick Vandercammen - One of the best experts on this subject based on the ideXlab platform.
-
binding of sorbitol 6 Phosphate and of Fructose 1 Phosphate to the regulatory protein of liver glucokinase
Biochemical Journal, 1992Co-Authors: Annick Vandercammen, Michel Detheux, E Van SchaftingenAbstract:Using a binding assay in which the ligand-protein complex is separated from free ligand by precipitation with poly(ethylene glycol) 6000, we found that the regulatory protein of rat liver glucokinase bound close to 1 mol of radiolabelled sorbitol 6-Phosphate, a negative effector, or of Fructose 1-Phosphate, a positive effector, per mol of regulatory protein. Scatchard plots were linear, the dissociation constant being 0.3 microM for both Phosphate esters. Sorbitol 6-Phosphate and Fructose 1-Phosphate competed with each other for the binding. Competition was also observed with psicose 1-Phosphate, ribitol 5-Phosphate, arabitol 5-Phosphate and 3-phosphoglycerate, all of which are known to affect the inhibition exerted by the regulatory protein. At a concentration of 10%, poly(ethylene glycol) 6000 decreased the concentration of regulatory protein causing 50% inhibition to a larger extent in the absence (12-fold) than in the presence (3-fold) of a saturating concentration of Fructose 6-Phosphate, another negative effector. Furthermore, it increased by about 3-fold the apparent affinity for inhibitory Phosphate esters, indicating that it induced conformational changes of the regulatory protein.
-
The regulatory protein of liver glucokinase
Advances in Enzyme Regulation, 1992Co-Authors: E Van Schaftingen, Annick Vandercammen, Michel Detheux, Dewi R. DaviesAbstract:Fructose, sorbitol and D-glyceraldehyde stimulate the rate of glucose phosphorylation in isolated hepatocytes. This effect is mediated by Fructose 1-Phosphate, which releases the inhibition exerted by a regulatory protein on liver glucokinase. In the presence of Fructose 6-Phosphate, the regulatory protein binds to, and inhibits, liver glucokinase. Fructose 1-Phosphate antagonizes this inhibition by causing dissociation of the glucokinase-regulatory protein complex. Both Phosphate esters act by binding to the regulatory protein, and by presumably causing changes in its conformation. The regulatory protein behaves as a fully competitive inhibitor. It inhibits liver glucokinase from various species, and rat islet glucokinase, but has no effect on hexokinases from mammalian tissues or from yeast, or on glucokinase from microorganisms. Kinetic studies indicate that the regulatory protein binds to glucokinase at a site distinct from the catalytic site. Several Phosphate esters, mainly polyol-Phosphates, were found to mimick the effect of Fructose 6-Phosphate. The most potent is sorbitol 6-Phosphate, suggesting that Fructose 6-Phosphate is recognized by the regulatory protein in its open-chain configuration. Other Phosphate esters and Pi have a Fructose 1-Phosphate-like effect. The stimulatory effect of Fructose on glucose phosphorylation is observed not only in isolated hepatocytes but also in the livers of anesthetized rats. This suggests that Fructose could be a nutritional signal causing an increase in the hepatic glucose uptake.
