Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.1 (hexokinase)
5,274 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

D-Glucosamine was found to be phosphorylated by a rat liver extract in the presence of a high concentration of glucose, which was formerly believed to be a strong competitive inhibitor of this reaction. Results suggested that glucosamine may be phosphorylated by high Km hexokinase, i.e. glucokinase [EC 2.7.1.2]. The enzyme involved was separated from specific N-acetyl-D-glucosamine kinase [EC 2.7.1.59]. The phosphorylation was not inhibited by a physiological level of glucose or glucose 6-phosphate, which strongly inhibited low Km hexokinase. The apparent Km of glucokinase for glucosamine was estimated as 8 mM, which is ten times that of low Km hexokinase.
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PMID:Phosphorylation of D-glucosamine by rat liver glucokinase. 115 56

We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus (1971, J. Biol. Chem. 246, 3885-3894) for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays.
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PMID:Radiometric assays for glycerol, glucose, and glycogen. 281 33

We have compared the competitive inhibitory effects of 2-deoxyglucose, glucosamine, N-acetylglucosamine, N-benzoylglucosamine, and the commonly used radiographic and density gradient agent metrizamide (2-[3-acetamido-2,4,6-triiodo-5-(N-methylacetamido) benzamido]-2-deoxyglucose) on the mitochondrial and soluble forms of human brain hexokinase. Metrizamide produces a classical competitive inhibition with glucose for human brain hexokinase, with Kis of 2.8 and 2.5 mM, respectively, for the mitochondrial and soluble forms. Glucosamine exhibited Kis of 0.58 and 0.29 mM, while 2-deoxyglucose exhibited Kis of 0.074 and 0.15 mM and N-acetylglucosamine 0.098 and 0.092 mM for these two forms, respectively. N-Benzoylglucosamine was by far the most effective inhibitor tested, with Ki values of 0.0086 and 0.022 mM, respectively. In order of increasing potency as a competitive inhibitor for mitochondrial hexokinase are metrizamide, glucosamine, N-acetylglucosamine, 2-deoxyglucose, and N-benzoylglucosamine. For the soluble form of the enzyme in increasing potency are metrizamide, glucosamine, 2-deoxyglucose, N-acetylglucosamine, and N-benzoylglucosamine. Since N-benzoylglucosamine was over 100 times more potent than metrizamide, some of the effects of metrizamide could be due to contamination by N-benzoylglucosamine. However, gas chromatography-mass spectrometry analysis of metrizamide did not indicate the presence of N-benzoyl-glucosamine. In addition, column chromatographic separation of commercially available metrizamide and reconstitution of freeze-dried eluate fractions localized the inhibitory effect to the metrizamide peak.
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PMID:Competitive inhibition of human brain hexokinase by metrizamide and related compounds. 669 84

Human beta-cell glucokinase recognition and phosphorylation of different sugars was investigated by steady-state kinetic analysis, measurements of substrate-induced intrinsic fluorescence changes, and molecular modeling and calculation of interaction energies. Measurements of kcat/Km showed that glucokinase phosphorylated the sugars in the order glucose = mannose > deoxyglucose > fructose = glucosamine. The mode of binding of these sugars to the open conformation of glucokinase was predicted from molecular modeling. Glucokinase is predicted to form similar interactions with the 6-OH, 4-OH, and 1-OH groups of all these sugars. The interactions of the 2-OH and 3-OH groups differ and depend on the type of sugar and reflect differences in cooperative behavior. For example, glucose and deoxyglucose exhibited cooperative behavior with Hill coefficients of 1.8 and 1.5, respectively, while mannose and fructose demonstrated Michaelis-Menten behavior. Galactose, allose, and 2,5-anhydroglucitol were not substrates under the assay conditions used, and the alpha- and beta-anomers of methylglucose were poor substrates with Km's greater than 1000 mM. Glucokinase exhibited an ATPase activity which was 1/2000th that of the rate of the kinase reaction, and unlike yeast hexokinase, it was not affected by the addition of lyxose. Glucosamine was a low affinity inhibitor as well as a substrate, while N-acetylglucosamine and mannoheptulose were high-affinity inhibitors. The change in intrinsic fluorescence that was induced by glucose, mannose, and mannoheptulose had the opposite sign for glucosamine, which implies a very different mode of binding from the other sugars. The calculated interaction energies of glucokinase with glucose, mannose, deoxyglucose, and fructose agree very well with the measured values of kcat/Km, which indicates that these sugars are recognized by binding to the open conformation of glucokinase.
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PMID:Sugar specificity of human beta-cell glucokinase: correlation of molecular models with kinetic measurements. 774 12

Glucosamine, a potent inhibitor of glucokinase (hexokinase IV or D), was used to estimate the contribution of this enzyme to glucose phosphorylation in freshly isolated rat hepatocytes and its sensitivity to fructose 6-phosphate in situ. Experiments with radiolabelled glucosamine indicated that this amino sugar, at concentrations of 5 or 40 mM, readily penetrated hepatocytes to reach in 1 min a total (i.e., glucosamine+metabolites) intracellular concentration equal to 0.8-1.2-fold its extracellular concentration. In marked contrast, N-acetylglucosamine barely penetrated the cells. The detritiation of [2-3H]glucose, used to estimate glucose phosphorylation in intact cells, was inhibited by glucosamine much more potently than by N-acetylglucosamine, half-maximal effects being reached at about 2.5 and 30 mM respectively. Extrapolation of the data indicated that about 12% of the detritiation was resistant to glucosamine. Dihydroxyacetone (10 mM), lactate (10 mM) + pyruvate (1 mM), and glucagon (1 microM) increased up to 8-fold the concentration of hexose 6-phosphates (glucose 6-phosphate+fructose 6-phosphate) and, against expectations, modestly decreased the detritiation rate measured in the absence of glucosamine. In the presence of 40 mM glucosamine, these agents increased the detritiation rate, which then positively correlated with the concentration of hexose 6-phosphates. This hexose 6-phosphates-dependent detritiation was sensitive to inhibition by vanadate, and was also catalysed by gel-filtered cell-free extracts, as well as by liver microsomes in the presence of phosphoglucoisomerase; it can be explained by an exchange reaction catalysed by glucose-6-phosphatase. When this exchange reaction is taken into account, it appears that the rate of glucose detritiation attributable to glucokinase decreases when the concentration of hexose 6-phosphates increases. This is in agreement with the known effect of fructose 6-phosphate to potentiate the inhibition of glucokinase by its regulatory protein.
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PMID:Glucosamine-sensitive and -insensitive detritiation of [2-3H]glucose in isolated rat hepatocytes: a study of the contributions of glucokinase and glucose-6-phosphatase. 775 69

N-Acetyl-D-[2-3H]glucosamine was synthesized from N-acetyl-D-mannosamine by alkaline 2-epimerization in pyridine containing 3H2O and nickelous acetate. The reaction involves reversible formation of an enol intermediate and therefore also resulted in incorporation of tritium into N-acetylmannosamine. After completed reaction, the two N-acetylhexosamines were separated from other radioactive products and Morgan-Elson chromogens by chromatography on a column of Sephadex G-10, which was eluted with 10% ethanol, and were then separated from each other by chromatography on Sephadex G-15 in 0.27 M sodium borate (pH 7.8). The location of the incorporated tritium was established by treatment of the N-acetylhexosamines with borate under the conditions of the Morgan-Elson reaction, which converts the sugars to Kuhn's chromogen I with concomitant loss of the C-2 hydrogen. As expected, this treatment resulted in the formation of 3H2O, indicating that the tritium was located at C-2. [2-3H]Glucosamine was prepared by acid hydrolysis of the labelled N-acetylglucosamine and was converted to [2-3H]glucosamine 6-phosphate by incubation with hexokinase and ATP. The sugar phosphate was used as a substrate for glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10) in a simple 3H2O release assay.
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PMID:Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water. 778 Jan 91

Glucosamine infusion induces insulin resistance in vivo, but the effect of glucosamine on intracellular metabolites of the hexosamine pathway, especially glucosamine-6-phosphate (GlcN6P) is unknown. Because of the structural similarity of glucose-6-phosphate (G-6-P) and GlcN6P, we hypothesized that accumulation of this metabolite might alter the activities of enzymes such as glycogen synthase and hexokinase. We infused glucosamine (30 micromol x kg(-1) x min(-1)) to induce insulin resistance in rats during a euglycemic-hyperinsulinemic clamp. Glucosamine induced whole-body insulin resistance, which was apparent after 90 min and continued progressively for 360 min. Despite inducing severe whole-body insulin resistance and decrease in glycogen synthase fractional activity in rectus abdominis muscle (69+/-3 vs. 83+/-1%, P<0.01) and heart (7+/-1 vs. 32+/-4%, P<0.001), glucosamine did not change the glycogen content in rectus and even increased it in the heart (209+/-13 vs. 117+/-9 mmol/kg dry wt, P<0.001). Glucosamine increased tissue concentrations of UDP-GlcNAc 4.4- and 4.6-fold in rectus abdominis and heart, respectively. However, GlcN6P concentrations increased 500- and 700-fold in glucosamine-infused animals in rectus abdominis (590+/-80 vs. 1.2+/-0.1 micromol/kg wet wt, P<0.001) and heart (7,703+/-993 vs. 11.2+/-2.3 micromol/kg wet wt, P<0.001). To assess the possible significance of GlcN6P accumulation, we measured the effect of GlcN6P on glycogen synthase and hexokinase activity in vitro. At the GlcN6P concentrations measured in rectus abdominis and heart in vivo, glycogen synthase was activated by 21 and 542%, while similar concentrations inhibited hexokinase activity by 5 and 46%, respectively. This study demonstrates that infusion of glucosamine during a euglycemic-hyperinsulinemic clamp results in marked accumulation of intracellular GlcN6P. The GlcN6P concentrations in the heart and rectus abdominis muscle reach levels sufficient to cause allosteric activation of glycogen synthase and inhibition of hexokinase.
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PMID:Allosteric regulation of glycogen synthase and hexokinase by glucosamine-6-phosphate during glucosamine-induced insulin resistance in skeletal muscle and heart. 1033 16

High-glucose exposure down-regulates protein kinaseC beta II posttranscriptionally in rat and human vascular smooth muscle cells and contributes to increased cell proliferation. High-glucose-induced mRNA destabilization is specific for PKC beta II mRNA, while PKC beta I and other PKC mRNA are not affected. This study focused on whether glucose metabolism was required. The effect was blocked by cytochalasin B, suggesting a requirement for glucose uptake. Glucosamine did not mimic the effect, indicating that metabolism via hexosamine pathway was not involved. The effect was hexokinase-independent since 3-O-methylglucose, in a dose-dependent manner, mimicked high-glucose effects. Cycloheximide did not block the effect excluding dependency on new protein synthesis. Wortmannin and LY294002, phosphoinositide 3-kinase (PI3-kinase) inhibitors, blocked glucose effects in the presence of 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole. Glucose and 3-O-methylglucose activated PI3-kinase, and LY294002 blocked glucose effects on Akt phosphorylation. In these cells, high-glucose concentrations activated a metabolically linked signaling pathway independent of glucose metabolism to regulate mRNA processing.
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PMID:Phosphoinositide 3-kinase mediates protein kinase C beta II mRNA destabilization in rat A10 smooth muscle cell cultures exposed to high glucose. 1206 8

Glucosamine (GlcN) is a naturally occurring amino-sugar that is synthesized by amidation of fructose-6-phosphate. Although a number of reports have examined the biological effects of GlcN on insulin resistance in mammalian systems, little is known about its effects on plant growth. In this study, we have shown that exogenous GlcN inhibits hypocotyl elongation in Arabidopsis, whereas glucose and its analogs alleviate this inhibitory effect. The hexokinase (HXK)-specific inhibitor mannoheptulose also restored hypocotyl elongation. The gin2-1 mutants with an alteration in AtHXK1 exhibited higher tolerance to GlcN. We also found that GlcN induces a significant increase in the production of reactive oxygen species (ROS). In addition, the GlcN-mediated inhibition of hypocotyl elongation was relieved by reducing agents such as ascorbic acid and glutathione. GlcN treatment resulted in significant induction of expression of GST1, GST2 and GST6, which are marker genes for ROS production. The gin2 mutation also represses the ROS production and the GST2 induction by GlcN treatment. Taken together, these results provide evidence that GlcN induces HXK-mediated induction of oxidative stress, leading to growth repression in Arabidopsis thaliana.
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PMID:Glucosamine causes overproduction of reactive oxygen species, leading to repression of hypocotyl elongation through a hexokinase-mediated mechanism in Arabidopsis. 1854 38

A series of five glucosamine-conjugated organometallic complexes of the tricarbonyl cores of technetium-99m and rhenium were made. Glucosamine was derivatized at the C-2 nitrogen with long chain alkyl spacers linked to either pyridyl-tert-nitrogen-phenol tridentate chelates or cyclopentadienyl ligating groups. The metal complexes of the tridentate ligands were formed by refluxing with [Re(CO)(3)(H(2)O)(3)]Br, or with a base and [(99m)Tc(CO)(3)(H(2)O)(3)](+). These ligands were found to be competent chelates in binding the [(99m)Tc(CO)(3)](+) core as radiolabeling yields ranged from 87 to 93% and the resulting complexes are stable to cysteine and histidine challenges for 24 h. The cyclopentadienyl analogues were formed using a double ligand transfer reaction for the rhenium complexes and a single ligand transfer for the technetium-99m complexes. All five rhenium complexes were tested as substrates of hexokinase; two of these complexes were tested as hexokinase inhibitors and they were found to be competent inhibitors, suggesting that they may be able to interact with hexokinase. MTT cytotoxicity studies were performed and the complexes tested were found to be non-toxic to the concentrations tested (100 microM or 1 mM). GLUT-1 mediated cell uptake studies were performed on all five technetium-99m complexes, and their cell entry was found to parallel their lipophilicities, suggesting that cellular uptake is by passive diffusion and is not mediated by GLUT-1.
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PMID:Long-chain rhenium and technetium glucosamine conjugates. 2044


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