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PubMedDate not supplied

Diurnal rhythms in the liver hexokinase and glucokinase activity, in the level of glycogen in liver, skeletal and heart muscles and in the level of blood glucose in laboratory mice.

The experiments were carried out on 84 male Swiss mice. The first group was kept in light-dark conditions L:D = 6:18 and the second one--in reversed photoperiod--L:D = 18:6. After 6 weeks of the experiment the level of glucose in blood, the activity of hexokinase and glucokinase, and the summary activity of these enzymes in the liver, the concentration of glycogen in the liver, myocardium and gastrocnemius muscle were determined. A diurnal rhythm in the level of blood glucose was observed in L:D = 6:18. The rhythm of hexokinase and glucokinase activity in the liver was detected neither under conditions of L:D = 6:18 nor in L:D = 18:6. A diurnal rhythm of the summary activity of hexokinase and glucokinase in the liver was detected in L:D = 6:18. The diurnal rhythm of the level of glycogen in the liver and gastrocnemius muscle was observed in both types of photoperiod. In the myocardium the rhythm was detected neither under L:D = 6:18 nor L:D = 18:6 conditions.

Animals↗

PubMed1978

[Relationship between the degree of the small intestine motility and the activity of hexokinase in its smooth muscle layer].

Interrelationship between the motility of the small intestine and the intensity of energy formation in its smooth muscle layer was studied. The hexokinase activity was found to be significantly higher in the muscle layer of the duodenum as compared to that activity in the ileum and jejunum. No statistically significant differences in the hexokinase activity were revealed between the ileum and jejunum. The results obtained showed hexokinase activity to be highly variable, these values directly correlating with the motor activity of the intestinal muscle layer, representing the contractile apparatus of the intestine.

Animals↗

PubMed1980

Multiple forms of Drosophila hexokinase. Purification, biochemical and immunological characterization.

Major forms of hexokinases (Hex A and Hex C) in Drosophila melanogaster were purified to homogeneity by utilizing affinity chromatography and preparative isoelectric focusing column as the key steps. Three different affinity ligands immobilized on Sepharose (3-amino pyridine adenine dinucleotide, glucosamine, and 8-(6-aminohexyl)-amino-ATP) were employed during different stages of enzyme purification. Antisera against purified Hex A and Hex C were raised in rabbits. Hex A, the major form in adults, and Hex B, the predominant form in larvae, showed complete immunological identity by double immunodiffusion and enzyme immunoinactivation studies. No cross-reactivity was observed between the antiserum to Hex A and Hex C or between the antiserum to Hex C and Hex A. By gel filtration chromatography and sodium dodecyl sulfate-acrylamide gel electrophoresis, all of the Drosophila hexokinases were shown to be monomers of molecular weights ranging from 40,000 to 50,000. Multiple forms of Drosophila hexokinase were studied extensively with respect to their biochemical properties including Michaelis constants, substrate and coenzyme specificities, pH-dependent activity, and thermal stability. Consistent with previous genetic evidence, the results of our studies also suggest that Hex A and Hex B (with subforms B1 and B2) are products of a single structural gene but are modified post-translationally, whereas the allelic forms of Hex C (C1 and C2) are derived from a different structural gene from that of Hex A and Hex B.

Animals↗

PubMed1983

Association equilibria and reacting enzyme gel filtration of yeast hexokinase.

The association-dissociation of yeast hexokinase has been re-examined and the size of the reacting form of the enzyme has been investigated under a variety of conditions. Both Sephacryl S-200 and high performance liquid chromatography on Bio-Sil have been employed with continuous effluent monitoring under reacting and nonreacting conditions. The reacting enzyme was monomeric under all conditions, suggesting that the dimer is not an important catalytic species in normal assays. The reacting enzyme remained as a monomer under the following conditions: 0.1-15 micrograms/ml loading concentration, from 30 to 100 mM ionic strength, with 2 mM citrate, with 50% D2O, and at 160 atm hydrostatic pressure. The dissociation constant for the nonreacting hexokinase was 22 (+/- 2) micrograms/ml (uncorrected for 5-fold dilution) in 100 mM triethanolamine, pH 6.5, and 25 degrees C. Glucose or MgATP had dissociative effects under all conditions studied, but MgATP was much less effective and only slightly more effective than an equivalent ionic strength. NaCl, between 30 and 80 mM, promoted dissociation, with a concomitant conformational change suggested by nonlinearity of log-log plots. The extent of dissociation with MgCl2 was slightly greater than an equivalent NaCl ionic strength and the shape of the association curves suggested the formation of an elongated dimer in the presence of MgCl2. The conclusion is made that hexokinase is monomeric under most assay conditions and that the dissociation is predominantly the result of the glucose interaction. High performance liquid chromatography has been shown to be a useful method of assessing the association state of enzymes under both reacting and nonreacting conditions.

Chromatography, Gel↗

PubMedDate not supplied

Action of orthophosphate on rabbit red blood cell hexokinase.

1. Orthophosphate (Pi) at low concentrations, is able to activate the glucose 6-phosphate, glucose-1,6-diphosphate or 2,3-diphosphoglycerate-inhibited hexokinase (E.C. 2.7.1.1). In contrast, at high concentrations it is competitive inhibitor of MgATP2- with a Ki of 20 mM. 2. The Pi effect on inhibited hexokinase is pH dependent and becomes lower as the pH increase. The similar pH-dependence of the Ki of glucose-1,6-diphosphate with the pH-independence of the Ki of glucose 6-phosphate here reported, suggest the coincidence of the C-1 phosphate, of glucose-1,6-diphosphate and Pi binding sites. 3. Activation of uninhibited hexokinase by Pi at pH lower than the physiological ones has been demonstrated to be an artefact.

Adenosine Triphosphate↗

PubMed1981

Localization of hexokinase in mitochondria from rabbit reticulocytes and its relation to mitochondrial ATP-formation studied by measurement of 32P-fluxes.

The submitochondrial distribution of hexokinase was studied by repeated specific solubilizations and by tryptic digestion of isolated rabbit reticulocyte mitochondria. Whereas most of the enzyme is dissociably bound to the outer side of outer mitochondrial membrane, a small tightly bound portion is localized more internally. Electrophoretic separations did not reveal a specific isoenzyme pattern of the internal mitochondrial enzyme. Relationships between mitochondrial hexokinases and the intramitochondrial ATP pool, generated by oxidative phosphorylation, were studied by measuring 32P-fluxes following gamma-32P-ATP pulses on phosphorylating and non-phosphorylating mitochondria. Under both conditions, the specific activities in deoxyglucose-6-phosphate correspond closely to that of total gamma-ATP, thus not supporting a preferential use of intramitochondrial generated ATP by part of the mitochondrial hexokinases.

Adenosine Triphosphate↗

PubMed1978

[Importance of binding of 2,3-diphosphoglycerate and ATP to hemoglobin for erythrocyte glycolysis: activation by 2,3-diphosphoglycerate of hexokinase at intracellular conditions].

In a theoretical study the influence of hemoglobin and Mg-ions as binding partners of red cell 2,3-diphosphoglycerate and ATP was investigated. Free hemoglobin may be an efficient competitor of Mg2+ for the ligand ATP. At conditions which favour hemoglobin as binding partner (i.e. desoxygenation, low medium pH and incubation temperature, as in blood preservation) up to 95% of the whole cellular ATP (ca. 2mM in cell water) may be bound to hemoglobin (ca. 7 mM). This binding is largely prevented in the presence of physiological amounts of diphosphoglycerate (ca. 7 mM) which is in excess and has a higher binding affinity to hemoglobin. Therefore, diphosphoglycerate keeps ATP (MgATP) in cell water solution at conditions in which Hb would trop it in the presence of Mg2+ (ca. 3mM). It can be calculated that, by lack of free MgATP, the activity of hexokinase within the cell drops by a factor of greater than 10 when diphosphoglycerate is metabolized. This indirect activation by diphosphoglycerate of hexokinase is operative at free concentrations of DPG far below those which exert the well known excess inhibitory effect on hexokinase and phosphofructokinase. In a model study, the activation by diphosphoglycerate of the initial two-kinase stage was introduced into a simplified kinetic model of glycolysis. A pronounced hysteresis loop of the stationary concentrations of ATP and diphosphoglycerate was produced indicating the existence of several stationary states, one with high ATP and high diphosphoglycerate, the other one with low values. It is demonstrated that diphosphoglycerate, being a protector of glycolysis at physiological concentrations, triggers an autocatalytic breakdown of the energy state when permitted to drop to low values.

Adenosine Triphosphate↗

PubMed1976

Interaction of tetraiodofluorescein with yeast hexokinase.

The dye tetraiodofluorescein (TIF) was found to be an effective inhibitor of yeast hexokinase. It is a competitive inhibitor relative to MgATP2- and a noncompetitive inhibitor of glucose binding, a kinetic pattern consistent with the previously proposed random kinetic mechanism. TIF interacts directly with the native dimeric protein to give a difference spectrum with a maximum at 543 nm. Monomeric protein (produced by addition of 0.6 M NaCl) interacts with TIF to give a slightly altered difference spectrum, with the gammamax at 545 nm. The difference spectrum of the dimeric form is not perturbed by the addition of substrates but the absorbance with the monomer is lowered by MgATP2-. The Kd for MgATP2- was estimated to be 7 nM for monomeric hexokinase. These results suggest that results of previous binding studies with hexokinase at high concentrations which have been interpreted as being at variance with kinetic studies are due likely to different conformations of the protein under different experimental conditions.

Adenosine Triphosphate↗

PubMed1994

Glucose transporters in single skeletal muscle fibers. Relationship to hexokinase and regulation by contractile activity.

Glucose transport and phosphorylation are the first steps in the utilization of extracellular glucose by skeletal muscle. We have examined the relationships between proteins mediating these steps in single fibers of identified type dissected from rabbit skeletal muscle. The level of the glucose transporter isoform GLUT4, measured by immunoblotting, varied among fibers by a factor of 20 (slow oxidative > fast oxidative glycolytic > fast glycolytic). In fibers from the tibialis anterior muscle, GLUT4 was correlated (r2 = 0.75) with the activity of malate dehydrogenase, an enzyme representative of oxidative energy metabolism. In these fibers a strong correlation (r2 = 0.70) was also observed between GLUT4 and hexokinase activity. GLUT1 levels were barely detectable, regardless of fiber type. To investigate the possible role of muscle activity in controlling the expression of transporters, tibialis anterior muscles were activated by chronic electrical stimulation of the peroneal nerves. GLUT1 levels increased after 1 day of stimulation to a plateau that was severalfold higher than the level in non-stimulated cells. Hexokinase activity and the GLUT4 level changed in parallel: both were increased by approximately 2.5-fold after 1 day and by 14-fold after 21 days. Thus, while both GLUT1 and GLUT4 were regulated by muscle activity, only GLUT4 expression was coordinated with the expression of hexokinase.

Animals↗

PubMed1994

Overexpression of hexokinase I but not GLUT1 glucose transporter alters concentration dependence of glucose-stimulated insulin secretion in pancreatic beta-cell line MIN6.

The recently established pancreatic beta-cell line MIN6 retains the ability to secrete insulin in response to physiological glucose concentrations. To investigate the role of glucose transport and phosphorylation in glucose-stimulated insulin secretion by beta-cells, MIN6 cells were stably transfected with a rabbit GLUT1 glucose transporter cDNA or a rat hexokinase I cDNA cloned in an expression vector. Overexpression of GLUT1 increased 3-O-methylglucose uptake, but did not alter either glucose utilization or glucose-stimulated insulin secretion. In contrast, clones overexpressing hexokinase I exhibited enhanced glucose-stimulated insulin secretion at glucose concentrations below 10 mM with a concomitant increase in glucose utilization. Maximal insulin secretion as well as the maximal rate of glucose utilization were not altered in these clones. Insulin secretion stimulated by 2-ketoisocaproate, a non-glucose secretagogue, was not affected by hexokinase I expression. These results strongly suggest that the glucose phosphorylating step, but not glucose transport step, regulates glucose-stimulated insulin secretion by modulating the glycolytic rate in the beta-cell.

3-O-Methylglucose↗

PubMed1993

Structure/function relationships in hexokinase. Site-directed mutational analyses and characterization of overexpressed fragments implicate different functions for the N- and C-terminal halves of the enzyme.

Hexokinases are comprised of two highly homologous approximately 50-kDa halves and are product-inhibited by glucose-6-P. Four amino acid residues, Ser603, Asp657, Glu708, and Glu742, located in the C-terminal half of the tumor mitochondrial enzyme have been shown to be essential for enzyme function (Arora, K. K., Filburn, C. R., and Pedersen, P. L. (1991) J. Biol. Chem. 266, 5359-5362). Here we have assessed also the role of the N-terminal half of the same enzyme. Site-directed mutagenesis of residues predicted to interact with glucose in the N-terminal half, i.e. Ser155, Asp209, and Glu260, to Ala, have no effect on hexokinase activity. In addition, inhibition by hexose mono- and bisphosphates is unchanged for each of the mutant enzymes. Significantly, the overexpressed N-terminal polypeptide is devoid of catalytic activity but does have the capacity to bind ATP-agarose and be released with ATP and glucose-6-P. In contrast, the overexpressed C-terminal polypeptide is catalytically active and shows the same product inhibition pattern as the complete 100-kDa parent enzyme. These results emphasize that the N-terminal half of tumor hexokinase is essential neither for catalysis nor product modulation. Rather, the N-terminal half may play another role, perhaps in modulation of the ATP/glucose-6-P-dependent binding of the enzyme to tumor mitochondria or by acting as a spacer between the outer mitochondrial membrane and the C-terminal catalytic unit.

Amino Acid Sequence↗

PubMed1997

Purification and characterisation of the hexokinase of Plasmodium berghei, a murine malaria parasite.

Hexokinase (EC 2.7.1.1) activity in cell-free Plasmodium berghei was 35 and 5 times higher as compared to normal and P. berghei-infected mouse erythrocytes, respectively. Maximal enzyme activity was present in the cytosolic fraction of the isolated parasite. Manifold purification of parasite hexokinase was achieved with Sephadex G-200. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed parasite enzyme subunit in the molecular weight range of 47 kDa with ATP (adenosine triphosphate) Km of 2 mM. Two out of three mice immunised with the hexokinase fraction were protected upon challenge with live parasites.

Adenosine Triphosphate↗

PubMed1997

Activation of hexokinase by mild insulin dependent diabetes mellitus in rat erythrocytes.

In order to reveal sequel of events responsible for increase in red cell cytosolic glucose-6 phosphate (G-6P) content of diabetic patients the enzyme producing and transforming G-6P were assayed. Increase in the activity of hexokinase and decrease in phosphoglucoisomerase activity was observed in mild insulin dependent diabetes mellitus (mIDDM) rat erythrocytes. Increase and decrease in activity of hexokinase and phosphoglucoisomerase respectively will increase the cytosolic glucose-6 phosphate content. Thus any substance which autoregulate the activity of hexokinase and maintains critical level of G-6P necessary for generation of ATP and coenzymes (NADPH & NAD+) in the prevailing hyperglycemic state can be a potential therapeutic agent for diabetic patients.

Animals↗

PubMedDate not supplied

[Change in the hexokinase activity of the rat myocardial and skeletal muscle mitochondria and cytoplasm in fasting and on a carbohydrate diet].

Dynamics of the hexokinase activity was stuided in mitochondria and cytoplasm of skeletal muscle and myocardium in rats which were subjected to fasting for three days and then were given a carbohydrate diet. It is established that the hexokinase activity in mitochondria (especially in the miocardium) lowers during fasting to a less extent as compared to cytoplasm and is not restored when feeding on carbohydrates, evidently, due to different quantitative ratio of isoenzymes. It is shown that hexokinase from the myocardium solubilizes more easily that from the skeletal muscles.

Animals↗

PubMed1990

Altered hexokinase activity in skin cultured fibroblasts and leukocytes from Alzheimer's disease patients.

Changes in the activity of brain glycolytic enzymes have been reported in Alzheimer's dementia. In this paper we studied the activity of the rate-limiting glycolytic enzymes, namely phosphofructokinase, lactate dehydrogenase and hexokinase in skin cultured fibroblasts and leukocytes from familial and sporadic Alzheimer's disease patients and unaffected relatives. Phosphofructokinase and lactate dehydrogenase activities were similar in all groups studied. The activity of hexokinase was reduced in some patients with the familial dominant form of Alzheimer's disease whilst it was normal in sporadic cases. These results suggest that Alzheimer's disease may be an heterogeneous disorder and that a modification on the catalytic activity of hexokinase may play a role in the pathogenesis of the disease in at least a subgroup of patients.

Alzheimer Disease↗

PubMed1999

Is hexokinase really a sugar sensor in plants?

The molecular mechanisms by which plant cells sense sugar levels are not understood, but current models (adapted from models for sugar sensing in yeast) favour hexokinase as the primary sugar sensor. However, the hypothesis that yeast hexokinase has a signalling function has not been supported by more recent studies and the idea that hexokinase is involved in sugar sensing in plants has yet to be proven.

Journal Article↗

PubMed1966

Kinetic studies with skeletal-muscle hexokinase.

Rat skeletal-muscle hexokinase was partially purified by ammonium sulphate fractionation and gel filtration. The mechanism of the skeletal-muscle hexokinase was studied kinetically by initial-velocity analysis and product inhibition. Glucose 6-phosphate was a non-competitive inhibitor of glucose and ATP. ADP was a non-competitive inhibitor of glucose and a competitive inhibitor of ATP. The data on product inhibition and initial-velocity analysis of skeletal-muscle hexokinase support an ordered sequential mechanism (ordered Bi Bi) where the addition of substrates and release of products is in the order: ATP, glucose, glucose 6-phosphate and ADP.

Journal Article↗

PubMed1976

The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is suggested that, under anaerobic conditions, muscles of marine invertebrates form lactate and/or octopine or succinate (or similar end product) according to the activities of the enzymes present in the muscles (see above). The muscles investigated possess low activities of cytosolic glycerol 3-phosphate dehydrogenase, which indicates that glycerol phosphate formation is quantitatively unimportant under anaerobic conditions, and low activities of mitochondrial glycerol phosphate dehydrogenase, which indicates that the glycerol phosphate cycle is unimportant in the re-oxidation of glycolytically produced NADH in these muscles under aerobic conditions. Conversely, high activities of glutamate-oxaloacetate transaminase are present in some muscles, which indicates that the malate-aspartate cycle may be important in oxidation of glycolytically produced NADH under aerobic conditions. 3. High activities of nucleoside diphosphate kinase were found in muscles that function for prolonged periods under anaerobic conditions (e.g...

Adenosine Triphosphate↗