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At least 613 records · Page 34Linked to original sources
PubMed2003

Metabolic profiling of transgenic tomato plants overexpressing hexokinase reveals that the influence of hexose phosphorylation diminishes during fruit development.

We have conducted a comprehensive metabolic profiling on tomato (Lycopersicon esculentum) leaf and developing fruit tissue using a recently established gas chromatography-mass spectrometry profiling protocol alongside conventional spectrophotometric and liquid chromatographic methodologies. Applying a combination of these techniques, we were able to identify in excess of 70 small-M(r) metabolites and to catalogue the metabolite composition of developing tomato fruit. In addition to comparing differences in metabolite content between source and sink tissues of the tomato plant and after the change in metabolite pool sizes through fruit development, we have assessed the influence of hexose phosphorylation through fruit development by analyzing transgenic plants constitutively overexpressing Arabidopsis hexokinase AtHXK1. Analysis of the total hexokinase activity in developing fruits revealed that both wild-type and transgenic fruits exhibit decreasing hexokinase activity with development but that the relative activity of the transgenic lines with respect to wild type increases with development. Conversely, both point-by-point and principal component analyses suggest that the metabolic phenotype of these lines becomes less distinct from wild type during development. In summary, the data presented in this paper demonstrate that the influence of hexose phosphorylation diminishes during fruit development and highlights the importance of greater temporal resolution of metabolism.

Arabidopsis↗
PubMed1999

Antisense repression of hexokinase 1 leads to an overaccumulation of starch in leaves of transgenic potato plants but not to significant changes in tuber carbohydrate metabolism.

Potato (Solanum tuberosum L.) plants transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 1 (StHK1) exhibited altered enzyme activities and expression of StHK1 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed a 22-fold variation in leaves (from 22% of the wild-type activity in antisense transformants to 485% activity in sense transformants) and a 7-fold variation in developing tubers (from 32% of the wild-type activity in antisense transformants to 222% activity in sense transformants). Despite the wide range of hexokinase activities, no change was found in the fresh weight yield, starch, sugar, or metabolite levels of transgenic tubers. However, there was a 3-fold increase in the starch content of leaves from the antisense transformants after the dark period. Starch accumulation at the end of the night period was correlated with a 2-fold increase of glucose and a decrease of sucrose content. These results provide strong support for the hypothesis that glucose is a primary product of transitory starch degradation and is the sugar that is exported to the cytosol at night to support sucrose biosynthesis.

Cell Line↗
PubMed2006

Mitochondria-associated hexokinases play a role in the control of programmed cell death in Nicotiana benthamiana.

Recent findings suggest a pivotal role for mitochondria-associated hexokinase in the regulation of apoptosis in animal cells. In this study, virus-induced gene silencing (VIGS) of a hexokinase-encoding Hxk1 caused necrotic lesions on leaves, abnormal leaf morphology, and retarded plant growth in Nicotiana benthamiana. Hxk1 was associated with the mitochondria, and this association required the N-terminal membrane anchor. VIGS of Hxk1 reduced the cellular glucose-phosphorylating activity to approximately 31% of control levels without changing the fructose-phosphorylating activity and did not alter hexose phosphate content severely. The affected cells showed programmed cell death (PCD) morphological markers, including nuclear condensation and DNA fragmentation. Similar to animal cell apoptosis, cytochrome c was released into the cytosol and caspase-9- and caspase-3-like proteolytic activities were strongly induced. Furthermore, based on flow cytometry, Arabidopsis thaliana plants overexpressing Arabidopsis HXK1 and HXK2, both of which are predominantly associated with mitochondria, exhibited enhanced resistance to H(2)O(2)- and alpha-picolinic acid-induced PCD. Finally, the addition of recombinant Hxk1 to mitochondria-enriched fractions prevented H(2)O(2)/clotrimazole-induced cytochrome c release and loss of mitochondrial membrane potential. Together, these results show that hexokinase critically regulates the execution of PCD in plant cells, suggesting a link between glucose metabolism and apoptosis.

Apoptosis↗
PubMed1999

Crystallization and preliminary crystal analysis of yeast hexokinase PI and PII.

Hexokinase is the prime enzyme of the Embden-Meyerhof pathway and is responsible for the first stage of energy conversion. It catalyzes the transfer of a phosphate to glucose to form glucose-6-phosphate. Yeast hexokinase PII is also known to play an important role in glucose signal transduction. Crystals of yeast hexokinase isoforms PI and PII were obtained by vapour-diffusion techniques using the hanging-drop method. Isoform PI crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 62.12, b = 78.87, c = 144.74 A. Unit-cell parameters for isoform PII crystals are a = b = 142.81, c = 58.46 A and the space group is I4. Synchrotron diffraction data have been collected to 2.2 A resolution from the isoform PII crystal, whereas isoform PI diffracted to 3.1 A.

Crystallization↗
PubMed1979

Affinity labelling of rat-muscle hexokinase type II by a glucose-derived alkylating agent.

The glucose-derived alkylating agent N-bromoacetylglucosamine (GlcNBrAc) is shown to cause a time-dependent irreversible inactivation of rat muscle hexokinase type II. The kinetics of inactivation are in accord with the reversible formation of an enzyme-inhibitor complex prior to modification, indicating that the reagent is active-site-directed. A Ki of 0.57 mM obtained for this reversible complexing is in agreement with a Ki of 0.65 mM obtained for the inhibition caused by N-propionylglucosamine, an isosteric analogue of GlcNBrAc and a competitive inhibitor with respect to glucose. Glucose itself protects competitively against inactivation. A KG of 0.26 mM obtained for the formation of enzyme-glucose complex from these studies is in agreement with the kinetically-determined Km of 0.2 mM. The substrate-unrelated but chemically similar alkylating agents bromoacetic acid and N-bromoacetylgalactosamine inactivate the enzyme at 20% of the rate caused by GlcNBrAc. The inactivation rate increases rapidly over the pH range 7--9. Analysis of this pH dependence shows that a single residue of pKa 8.9 is reacting with GlcNBrAc with a kmax (pH corrected, pseudo-first-order rate constant) of 1.5 x 10(-3) S-1. These values are typical of the reaction of model thiols with alkylating agents and suggests the reacting residue is probably a cysteine. Use of radioactively labelled GlcNBrAc indicates that uptake of 1 mol of reagent per mol protein causes complete activity loss. Finally the behaviour of this enzyme with active-site-directed alkylating agents is compared with published results of similar experiments carried out with yeast hexokinase and bovine brain hexokinase type I.

Acetylglucosamine↗
PubMed1984

Suppression of kinetic cooperativity of hexokinase D (glucokinase) by competitive inhibitors. A slow transition model.

Hexokinase D ('glucokinase') displays positive cooperativity with mannose with the same h values (1.5-1.6) as with glucose but with higher K0.5 values (8 mM at pH 8.0 and 12 mM at pH 7.5). In contrast, fructose and 2-deoxyglucose exhibit Michaelian kinetics [Cárdenas, M. L., Rabajille, E., and Niemeyer, H. (1979) Arch. Biol. Med. Exp. 12, 571-580; Cárdenas, M. L., Rabajille, E., and Niemeyer, H. (1984) Biochem. J. 222, 363-370]. Mannose, fructose, 2-deoxyglucose and N-acetylglucosamine acted as competitive inhibitors of glucose phosphorylation and decreased the cooperativity with glucose. Their relative efficiency for reducing the value of h to 1.0 was: fructose greater than mannose greater than 2-deoxyglucose greater than N-acetylglucosamine. Galactose, which is not a substrate nor an inhibitor, was unable to change the cooperativity. The competitive inhibition of glucose phosphorylation by N-acetylglucosamine or mannose was cooperative at very low glucose concentrations (less than 0.5 K0.5), suggesting the interaction of the inhibitors with more than one enzyme form. These and previously reported results are discussed on the basis of a slow transition model, which assumes that hexokinase D exists mainly in one conformation state (E1) in the absence of ligands and that the binding of glucose (or mannose) induces a conformational transition to EII. This new conformation would have a higher affinity for the sugar substrates and a higher catalytic activity than EI. Cooperativity would emerge from shifts of the steady-state distribution between the two enzyme forms as the sugar concentration increase. The inhibitors would suppress cooperativity with glucose by inducing or trapping the EII conformation. In addition, the model postulates that the different kinetic behaviour of hexokinase D with the different sugar substrates, cooperative with glucose and mannose and Michaelian with 2-deoxyglucose and fructose, is the consequence of differences in the velocities of the conformational transitions induced by the sugar substrates.

Acetylglucosamine↗
PubMed1990

Phosphorylation of yeast hexokinases.

We show by the use of 32P-labeling in vivo that hexokinase 2 and hexokinase 1 in Saccharomyces cerevisiae are phosphoproteins. The highest labeling was after incubation in medium with a low concentration of glucose, when labeling appears to be predominant even without use of immunoprecipitation. The nature of the modification is not known, but it has properties consistent with a phosphomonoester of serine or threonine. The cAMP-dependent protein kinase plays a negative role in hexokinase phosphorylation, in that there was reduced labeling in strains (bcy1) lacking a regulatory subunit, and increased labeling during growth with high concentrations of glucose in a strain attenuated in the catalytic subunit (tpk1w1). The function of the modification is not known, but there was a correlation between the extent of labeling and the expression of kinase-dependent high-affinity glucose uptake.

Cyclic AMP↗
PubMedDate not supplied

Kinetic properties of hexokinase of germ cells in rat testis.

The Michaelis-Menten constants (Km) of glucose, fructose, galactose, 2-deoxy-D-glucose and ATP as substrate for hexokinase of spermatocyte, spermatid and cauda epididymal spermatozoa extracts were measured. The Km value of glucose was very similar for all three germ cells. It was also true for all other substrates. The affinity of glucose for this enzyme was the highest while that of fructose was the lowest. The Km values were in general agreement with characteristics of hexokinase extracted from other tissues of rat. The Vmax values were also determined. The Vmax ratio of fructose to glucose showed the highest values of 1.1. The Vmax ratios of other substrates to glucose were below 1.0. The results suggest that hexokinase in the germ cells is similar to that in other tissues in its kinetic properties.

Animals↗
PubMed1983

Changes in hexokinase isoenzymes in regions of rat brain during insulin-induced hypoglycemia.

Activities of hexokinase isoenzymes were determined during insulin-induced hypoglycemia in soluble and total particulate fractions from three regions of rat brain. Type I hexokinase isoenzyme activity showed a small decrease in both soluble and particulate fractions from the cerebral hemispheres. In cerebellum and brain stem, however, Type I isoenzyme showed a decrease only in the soluble fraction. A significant increase was observed in hexokinase Type II isoenzyme from both the fractions, in all the three brain regions 1 h after insulin administration.

Animals↗
PubMed1983

The identity of hexokinase activities from mitochondrial and cytoplasmic fractions of rat brain homogenates.

Cytoplasmic hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified from the soluble fraction of a rat brain homogenate by a procedure that included a unique affinity elution of the enzyme from Blue Dextran-Sepharose. The purified enzyme was examined with respect to properties in which the impure cytoplasmic enzyme has been reported to differ from the solubilized mitochondrial enzyme. These included the ability to bind to mitochondria, inhibition by quercetin, effect of pH on activity, and kinetics. In all regards the purified mitochondrial and cytoplasmic enzymes appeared identical. In addition, comparative peptide maps after partial proteolysis showed no detectable differences. These results do not support the view that there exist distinct mitochondrial and cytoplasmic forms of hexokinase, the latter being permanently relegated to a cytoplasmic location and unable to participate in a dynamic equilibrium with the mitochondrially-bound enzyme. Alternatives are proposed to explain previous results that had been interpreted as indirect evidence for the existence of a distinct cytoplasmic hexokinase.

Animals↗
PubMed1983

The influence of specific phospholipids on the interaction of hexokinase with the outer mitochondrial membrane.

Repeated washing of a brain mitochondrial fraction results in a progressive decrease in the proportion of mitochondrially bound hexokinase that can be solubilized during a subsequent incubation with glucose-6-phosphate (glucose-6-P). Phospholipids removed during the washing procedure can be added back to washed mitochondria, resulting in enhancement of the solubilization by glucose-6-P. Column and thin-layer chromatographic methods have been used to isolate and identify active phospholipids. Additional studies were performed with purified lipids obtained commercially. Both lysophospholipids and acidic phospholipids were active in enhancing solubilization of hexokinase by glucose-6-P. Phospho-inositides, particularly diphosphoinositide, were quite effective, raising the possibility that the actively metabolized phosphoinositides may be involved in regulation of hexokinase binding in vivo.

Animals↗
PubMed1984

Competitive inhibition of human brain hexokinase by metrizamide and related compounds.

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.

Adult↗
PubMed1994

Hexokinase kinetics in human skeletal muscle after hyperinsulinaemia, hyperglycaemia and hyperepinephrinaemia.

The effects of 120 min of euglycaemic hyperinsulinaemia (UH, approximately 5 mM; 40 mU m-2 min-1), UH plus adrenaline infusion (0.05 microgram kg-1 min-1), and hyperglycaemic normoinsulinaemia (26 mM) on hexokinase kinetics in human skeletal muscle were examined. Biopsies were obtained from the quadriceps femoris muscle before and after each clamp. Total muscle hexokinase activity (HKt) (measured on a 2500 g supernatant) at a saturating level of the substrate glucose (1 mM) averaged 13 mmol kg dry wt-1 min-1 in the basal state and did not change significantly under any condition. Soluble hexokinase activity (HKs) (16,000 g supernatant) accounted for approximately 65% of HKt in the basal state, and this percentage was not significantly affected by any condition, suggesting that there was no major transfer of HK between cytosol and mitochondria. The activity of HKt and HKs was inhibited by glucose 1,6-bisphosphate (G-1,6-P2) in a concentration dependent manner in the basal state, and the sensitivity to G1,6-P2 inhibition was not altered by any condition. The activity of HKt and HKs in the presence of a subsaturating level of glucose (0.1 mM) accounted for approximately 70% of the activity at 1 mM glucose, and this percentage was not altered by any condition. These data suggest that under the present conditions alterations in the rates of whole body glucose disposal cannot be associated with alterations in HK distribution between cellular compartments nor its measured kinetics properties.

Epinephrine↗
PubMed1967

Testis-specific and sex-associated hexokinases in Drosophila melanogaster.

Hexokinases of Drosophila melanogaster were investigated by starchgel electrophoresis. A hexokinase is present in both sexes during earlier stages of development, but it persists only in male adults. In addition, in this species there is a testis-specific hexokinase which is first observed during the pupal period.

Animals↗
PubMed1968

17 beta-estradiol: inducer of uterine hexokinase.

Administration of estradiol to ovariectomized rats induced new synthesis of uterine hexokinase which was prevented by actinomycin D, 5-fluorouracil, cycloheximide, or ethionine. The estradiol-induced increase in uterine hexokinase activity was detectable as early as 4 hours. The increase in enzyme activity was dependent upon the dose of the hormone. The evidence indicates that the increased hexokinase activity induced by 17beta-estradiol may entail an acceleration of the synthesis of certain RNA species.

Animals↗
PubMed2001

Physiological properties of Saccharomyces cerevisiae from which hexokinase II has been deleted.

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The hxk2 mutant had a higher mitochondrial H(+)-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2 mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.

Aerobiosis↗
PubMed1998

Carbon source-dependent phosphorylation of hexokinase PII and its role in the glucose-signaling response in yeast.

The HXK2 gene is required for a variety of regulatory effects leading to an adaptation for fermentative metabolism in Saccharomyces cerevisiae. However, the molecular basis of the specific role of Hxk2p in these effects is still unclear. One important feature in order to understand the physiological function of hexokinase PH is that it is a phosphoprotein, since protein phosphorylation is essential in most metabolic signal transductions in eukaryotic cells. Here we show that Hxk2p exists in vivo in a dimeric-monomeric equilibrium which is affected by phosphorylation. Only the monomeric form appears phosphorylated, whereas the dimer does not. The reversible phosphorylation of Hxk2p is carbon source dependent, being more extensive on poor carbon sources such as galactose, raffinose, and ethanol. In vivo dephosphorylation of Hxk2p is promoted after addition of glucose. This effect is absent in glucose repression mutants cat80/grr1, hex2/reg1, and cid1/glc7. Treatment of a glucose crude extract from cid1-226 (glc7-T152K) mutant cells with lambda-phosphatase drastically reduces the presence of phosphoprotein, suggesting that CID1/GLC7 phosphatase together with its regulatory HEX2/REG1 subunit are involved in the dephosphorylation of the Hxk2p monomer. An HXK2 mutation encoding a serine-to-alanine change at position 15 [HXK2 (S15A)] was to clarify the in vivo function of the phosphorylation of hexokinase PII. In this mutant, where the Hxk2 protein is unable to undergo phosphorylation, the cells could not provide glucose repression of invertase. Glucose induction of HXT gene expression is also affected in cells expressing the mutated enzyme. Although we cannot rule out a defect in the metabolic state of the cell as the origin of these phenomena, our results suggest that the phosphorylation of hexokinase is essential in vivo for glucose signal transduction.

Adaptation, Biological↗
PubMed1998

Regulation of hexokinase II activity and expression in human muscle by moderate exercise.

A single bout of exercise increases the rate of insulin-stimulated glucose uptake and metabolism in skeletal muscle. Exercise also increases insulin-stimulated glucose 6-phosphate in skeletal muscle, suggesting that exercise increases hexokinase activity. Within 3 h, exercise increases hexokinase II (HK II) mRNA and activity in skeletal muscle from rats. It is not known, however, if a single bout of moderate-intensity exercise increases HK II expression in humans. The present study was undertaken to answer this question. Six subjects had percutaneous biopsies of the vastus lateralis muscle before and 3 h after a single 3-h session of moderate-intensity aerobic (60% of maximal oxygen consumption) exercise. Glycogen synthase, HK I, and HK II activities as well as HK I and HK II mRNA content were determined from the muscle biopsy specimens. The fractional velocity of glycogen synthase was increased by 446 +/- 84% after exercise (P < 0.005). Hexokinase II activity in the soluble fraction of the homogenates increased from 1.2 +/- 0.4 to 4.5 +/- 1.6 pmol.min-1.microgram-1 (P < 0.05) but was unchanged in the particulate fraction (4.3 +/- 1.3 vs. 5.3 +/- 1.5). HK I activity in neither the soluble nor particulate fraction changed after exercise. Relative to a 28S rRNA control signal, HK II mRNA increased from 0.091 +/- 0.02 to 0.195 +/- 0.037 (P < 0.05), whereas HK I mRNA was unchanged (0.414 +/- 0.061 vs. 0.498 +/- 0.134, P < 0.20). The increase in HK II activity after moderate exercise in healthy subjects could be one factor responsible for the enhanced rate of insulin-stimulated glucose uptake seen after exercise.

Adult↗