Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “HEXOKINASE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 757 records · Page 42Linked to original sources
PubMed1998

Clotrimazole and bifonazole detach hexokinase from mitochondria of melanoma cells.

Cancer cells are characterized by a high rate of glycolysis. Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), the only glycolytic enzyme which binds to mitochondria, is exceptionally high in cancer cells, and believed to play a key role in regulating cell energy metabolism and cancer cell growth rate. We have previously found that clotrimazole (1-(alpha-2-chlorotrityl)imidazole) and bifonazole (1-(alpha-biphenyl-4-ylbenzyl)imidazole), the antifungal azole derivatives, which were recently recognized as calmodulin antagonists, are calmodulin antagonists which most effectively reduce glycolysis and ATP level in B16 melanoma cells. They act through allosteric regulation and detachment of glycolytic enzymes from cytoskeleton. Here we report of a novel, additional, mechanism of action of these drugs. We show that they induce a dose-dependent detachment of hexokinase from mitochondria of B16 melanoma cells. This effect preceded the decrease in cell viability. These results suggest that clotrimazole and bifonazole may be promising drugs in treatment of melanoma.

Animals↗
PubMed2000

In vivo measurements of control coefficients for hexokinase and glucose-6-phosphate dehydrogenase in Xenopus laevis oocytes.

Hexokinase and glucose-6-phosphate dehydrogenase activities were increased in Xenopus laevis oocytes by microinjection of commercial pure enzymes. The effect of increased fractional activities on glycogen synthesis or on the production of 14CO(2) (the oxidative portion of the pentose phosphate pathway) was investigated by microinjection of [1-(14)C]glucose and measurements of the radioactivity in glycogen and CO(2). Control coefficients calculated from the data show that hexokinase plays an important role in the control of glycogen synthesis (control coefficient=0.7) but its influence on the control of the pentose phosphate pathway is almost nil (control coefficient=-0.01). Glucose-6-phosphate dehydrogenase injections did not affect the production of 14CO(2) by the pentose phosphate pathway, indicating that other factors control the operation of this pathway. In addition, an almost null control of this enzyme on glycogen synthesis flux was observed.

Animals↗
PubMed1998

Hexokinase PII has a double cytosolic-nuclear localisation in Saccharomyces cerevisiae.

We describe here that the HXK2 gene product, isoenzyme PII of hexokinase, is located in both the nucleus and the cytoplasm of Saccharomyces cerevisiae cells. This conclusion is supported by assays of hexokinase-specific activity in isolated nuclei from wild-type and hxk1lhxk2 double mutant strains, by immunoblot experiments using anti-Hxk2 antibodies and by observation of the fluorescence distribution of a Hxk2-GFP fusion protein in cells transformed with the HXK2::gfp gene.

Cell Fractionation↗
PubMed1998

The hexokinase 2 protein participates in regulatory DNA-protein complexes necessary for glucose repression of the SUC2 gene in Saccharomyces cerevisiae.

The HXK2 gene plays an important role in glucose repression in the yeast Saccharomyces cerevisiae. Recently we have described that the HXK2 gene product, isoenzyme 2 of hexokinase, is located both in the nucleus and in the cytoplasm of S. cerevisiae cells. In this work we used deletion analysis to identify the essential part of the protein-mediating nuclear localisation. Determinations of fructose-kinase activity and immunoblot analysis using anti-Hxk2 antibodies in isolated nuclei, together with observations of the fluorescence distribution of Hxk2-GFP fusion protein in cells transformed with an HXK2::gfp mutant gene, indicated that the decapeptide KKPQARKGSM, located between amino acid residues 7 and 16 of hexokinase 2, is important for nuclear localisation of the protein. Further experimental evidence, measuring invertase activity in wild-type and mutant cells expressing a truncated version of the Hxk2 protein unable to enter the nucleus, shows that a nuclear localisation of Hxk2 is necessary for glucose repression signalling of the SUC2 gene. Furthermore, we demonstrate using gel mobility shift analysis that Hxk2 participates in DNA-protein complexes with cis-acting regulatory elements of the SUC2 gene promoter.

Amino Acid Sequence↗
PubMed1992

Local probes and heterogeneous catalysis: a case study of a mitochondria-luciferase-hexokinase coupled system.

Biological systems are characterized by a high degree of structural organization. In the intracellular context, this introduces physical constraints which are not considered in the standard biochemical analysis of isolated systems, aimed towards mechanistic studies. A major challenge in cellular biology is thus to integrate the structural and mechanistic information and reach an adequate representation of the modes of operation in situ. We present an approach to this problem which takes advantage of a localized probe to study heterogeneous coupled system, as minimal models for cellular operation. The system consists of ATP production at the surface of mitochondria, and ATP consumption in solution by the hexokinase reaction. Soluble or biologically localized firefly luciferase is used to continuously monitor ATP concentration either in the bulk solution or at the surface of the organelle, respectively. The general system of a surface source and a bulk sink is mathematically modeled, and an analytic steady-state solution for local and bulk ATP is presented. The results are validated by experiment and differ from the expected behavior of an equivalent homogeneous system in solution. The model is further adapted to evaluate the effect of mixing. In addition, two limiting cases of heterogeneous distribution of hexokinase are analyzed, in which the soluble enzyme adsorbs non-specifically to mitochondria, or binds selectively to the site of ATP appearance on the membrane. The results are discussed in terms of their significance to the analysis of bulk measurements in vitro and their relevance to better description of cellular situations.

Adenosine Triphosphate↗
PubMed1982

Effects of divalent metal ions on the fluorescence and glucose-quenching of yeast hexokinase isozymes.

Titrations of the quenching of the tryptophan fluorescence of yeast hexokinase isozymes P-I and P-II by Mg2+, Mn2+, Ca2+, Cd2+, and Zn2+ ions and by glucose in the presence of each of these ions (10mM) were performed at pH 5.5 and 6.5 at 20 degrees C. At the higher pH there was a reversal of the type of glucose-binding cooperativity for P-II from negative to positive when either Mn2+ or Ca2+ was present in the buffered isozyme solution before the glucose titration, whereas Mg2+ caused the glucose binding to become noncooperative. Zn2+ and Cd2+ decreased the glucose quenching of P-II fluorescence drastically at pH 5.5, from a value of 15% in buffer to only 4%. Thus, only these two ions, of the five studied, cause the conformation change that results in quenching of the glucose-quenchable cleft tryptophan of P-II. Glucose binding to the P-I isozyme exhibited positive cooperativity in the presence of either Ca2+, Mg2+, or Mn2+, as well as in buffer alone, at both pH's. At the lower pH, Ca2+ enhanced the efficiency of glucose quenching of P-I fluorescence several-fold, while Mn2+ increased it only about 40% and Mg2+ not at all. Further, Ca2+ raised the degree of cooperativity (Hill coefficient) of glucose binding to P-I at this pH from the value of 1.42 in buffer and in the presence of Mg2+ and Mn2+ to 1.94, i.e., almost up to the highest possible value, 2, for dimeric hexokinase. However, at pH 6.5 the Ca2+ effect on the cooperativity was negligible, while Mg2+ and Mn2+ decreased the coefficient from 1.6 in buffer to about 1.4. The biological implications of these diverse metal ion effects are discussed.

Cations, Divalent↗
PubMed1998

Expression of hexokinase mRNA in human hippocampus.

The expression of hexokinase messenger RNA was evaluated in human hippocampus using in situ hybridization technique. The message showed an uneven distribution with high levels present in the granular cell layer of the dentate gyrus and CA3 region. The detection of specific transcripts was also observed in the lateral geniculate nucleus, the dentate polymorphic cell layer and the parahippocampal gyrus. The data suggest that, in the hippocampus, the expression of hexokinase is higher in neurons than in glial cells and that the rate of glucose metabolism may display considerable variations in the different subregions of this area.

Adult↗
PubMed1997

Effect of temperature on the activities of glucose-6-phosphate dehydrogenase and hexokinase in entomopathogenic nematodes (Nematoda: Steinernematidae).

The kinetic properties of two metabolic enzymes, glucose-6-phosphate dehydrogenase and hexokinase, were studied in four strains of entomopathogenic nematodes that had been recycled for two years at various temperatures: Steinernema feltiae NF strain, Steinernema feltiae Umeå strain, Steinernema carpocapsae All strain, Steinernema riobravis TX strain. The recycling temperatures influenced the activities of glucose-6-phosphate dehydrogenase and hexokinase in an adaptive fashion in all the strains. At each assay temperature (5-35 degrees C), the maximum specific activity of both the enzymes was greater in the nematodes that had been recycled at lower temperatures than in those reared at higher temperatures. In three enzyme-nematode strain combinations, the lowest K(m) values measured at each assay temperature occurred in nematodes that had been recycled at the lower temperatures. However, the assay temperatures at which the minimum K(m) values occurred were > or = 15 degrees C. The capacities of these nematodes to adjust to different recycling temperatures is discussed in relation to the physiological mechanisms involved.

Animals↗
PubMed2000

Hexokinase I is a Gli2-responsive gene expressed in the embryonic CNS.

Little is known about the downstream genes regulated by Gli zinc finger transcription factors, which are targets and mediators of Hedgehog signaling. Specifically, the identity and regulation of genes which mediate Gli2 function in neurogenesis are unclear. We describe here the cloning of frog Hexokinase I (HKI) as a Gli2-responsive gene. We show that HKI expression is induced by Gli2 and that it is detected in defined neuronal populations. Since the primary energy source of the brain is derived from glucose metabolism and hexokinase catalyses the first and rate limiting step in this process, the conversion of glucose into glucose-6-phosphate, these findings suggest a link between the regulation of neuronal induction and differentiation.

Amino Acid Sequence↗
PubMed2000

Time-resolved fluorescence of tryptophans in yeast hexokinase-PI: effect of subunit dimerization and ligand binding.

Time-resolved and steady-state fluorescence measurements have been performed on monomeric and dimeric forms of yeast hexokinase-PI. Observation of similar emission spectra and fluorescence decay parameters for both the forms of the enzyme suggests that tryptophan residue(s) are not likely to be present at the subunit-subunit interface and the process of dimerization does not perturb the local environment of tryptophan(s). The fluorescence decay of tryptophans in enzyme could be fitted to a bi-exponential function with two lifetime components, tau1 approximately 2.2 ns and tau2 approximately 3.9 ns. Binding of glucose, which is known to convert the 'open' conformation of the enzyme to a 'closed' active conformation, results in approximately 30% reduction in emission intensity and a selective decrease in tau1 from approximately 2.2 to approximately 1.1 ns. These effects can be reversed by the addition of trehalose 6-phosphate (an inhibitor of yeast hexokinase), suggesting that the trehalose 6-phosphate inhibits the enzyme by binding to its 'open' inactive conformation rather than competing with glucose to bind to the 'closed' active conformation. Binding of nucleotide ligands (ATP, ADP and adenyl-(beta,gamma-methylene)-diphosphate (AMPPCP)) to the monomeric or dimeric form of enzyme quenched the steady-state fluorescence by approximately 4-8%, but had no measurable effect on the distribution of lifetimes or on their magnitudes. Addition of nucleotides to the enzyme-glucose complex also did not produce any further change in fluorescence decay parameters. These results indicate that it is highly unlikely that the formation of a ternary enzyme-glucose-nucleotide complex from the binary enzyme-glucose complex is accompanied by a large conformational change in the enzyme, as has been surmised in some earlier studies.

Dimerization↗
PubMed1998

Interaction of bis(1-anilino-8-naphthalenesulfonate) with yeast hexokinase: a steady-state fluorescence study.

Bis(1-analino-8-naphthalenesulfonate) (bis-ANS) is a useful probe for hydrophobic areas on protein molecules and it has been proposed that it has a general affinity for the nucleotide binding site(s). There appear to be two different classes of binding sites for bis-ANS on hexokinase and these can be tentatively assigned as primary and secondary binding sites. The rate of binding of bis-ANS at the primary binding site is fast, whereas binding at secondary site(s) is slow. The slow increase in the fluorescence intensity on binding with bis-ANS is not due to conformational change in the enzyme, which may lead to the increase in the quantum yield of the bound dye. Circular dichroism measurements indicate that there is no significant change in the secondary structure on binding with this probe. In the presence of saturating amounts of glucose, the increase in fluorescence intensity due to binding at the secondary binding site(s) is significantly lowered. This indicates that glucose-induced conformational change has been sensed by this probe. From kinetic studies, it has been observed that bis-ANS is an effective competitive inhibitor of yeast hexokinase with respect to ATP. The stoichiometry of binding of this fluorescent probe is about one per subunit at the primary site both in the presence and absence of glucose, and the dissociation constant of bis-ANS is unaffected by glucose. It is possible to decrease significantly the amount of fluorescence intensity at the primary site by nucleotides. These results indicate that bis-ANS interacts at the site where nucleotide interacts. Energy transfer experiments indicate the proximity of some tryptophan(s) and bound bis-ANS molecule(s).

Adenosine Diphosphate↗
PubMed2003

Lithium detaches hexokinase from mitochondria and inhibits proliferation of B16 melanoma cells.

Glycolysis is known to be the primary energy source in cancer cells. Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), the only glycolytic enzyme which binds to mitochondria, is exceptionally high in cancer cells, and believed to play a key role in regulating cell energy metabolism and cancer cell growth rate. We show here that lithium induces a detachment of hexokinase from mitochondria of B16 melanoma cells. This effect eventually leads to inhibition of cell proliferation. These results reveal a novel, additional, mechanism of action of lithium and suggest that lithium may be promising drug in treatment of melanoma.

Animals↗
PubMed2000

Long chain fatty acids inhibit and medium chain fatty acids activate mammalian cardiac hexokinase.

We investigated the effect of non-esterified fatty acids (FAs) on bovine heart hexokinase (type I: ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). Long chain FAs (C14 to C20) inhibited the enzyme in a way that correlated positively with both the chain length and the degree of unsaturation. Medium chain FA with 12 or less carbons activated hexokinase in a chain length dependent manner with the greater activation shown by laurate. The activation constant of laurate was 91.5 microM with a maximal activation of 60.3%. Oleate caused a maximal decrease in specific activity of 25% with an inhibition constant of 79 microM. Using the fluorescent probe cis-parinarate, we found a saturable binding site with K(d) of 3.5 microM. Oleate competed the fluorescent probe from the protein with a K(d) of 1.4 microM. Medium chain FAs did not compete the probe from HK. The binding of fatty acid to the protein appears to be entropically driven as indicated by an Arrhenius analysis (DeltaS=+231.6 J mol(-1) deg(-1)). The presence of oleate significantly increased the K(ATP)(m) from 0.47 mM to 0.89 mM while the K(glucose)(m) in the presence of the FA (0.026+/-0.003 mM) was not significantly different from the control (0.014+/-0.004 mM). A decrease in V(max) values in the presence of oleate indicated that a mixed allosteric inhibition was operating.

Adenosine Triphosphate↗
PubMedDate not supplied

Synthesis and in vitro characterization of organometallic rhenium and technetium glucose complexes against Glut 1 and hexokinase.

A series of nine organometallic technetium-99m and rhenium complexes of glucose are presented and characterized in vitro regarding their potential as surrogates of [18F]-2-fluoro-desoxy glucose ([18F]-FDG). The glucose derivatives are functionalized at positions C-1, C-2, C-3, and C-6. Different spacer lengths and chelating systems have been introduced at these sites. For the (radio)labeling, the organometallic precursors [99mTc(H2O)3(CO)3]+ and [ReBr3(CO)3](2-) respectively have been used. The resulting complexes have been characterized chemically and radiochemically. The formation of uniform products has been observed on the macroscopic (Re) and no-carrier-added level (99mTc). The Tc-99m complexes revealed good inertness against ligand exchange (Cys and His) and excellent stability in physiological buffered saline as well as in human plasma over a period of 24 h at 37 degrees C. The rhenium complexes have been tested for competitive inhibition of the (yeast) hexokinase. Only for C-2 derivatized glucose complexes with extended spacer functionalities Ki values in the millimolar and sub-millimolar range have been observed. In silico molecular docking experiments supported these experimental findings. However, the competitive inhibitors are not recognized as a pseudosubstrate of hexokinase. The cellular uptake of all 99mTc-complexes into HT-29 colon carcinoma cells via Glut1 was generally low and unspecific independent of the position at the hexose ring, the chelating systems, or the overall charge of the corresponding metal complexes. The current results seem to preclude the use of these compounds as [18F]-FDG surrogates primarily due to the low cellular uptake via Glut1.

Binding, Competitive↗
PubMed1983

Inactivation of yeast hexokinase B by triethyltin bromide.

Triethyltin bromide was found to demonstrate temperature-dependent inactivation of yeast hexokinase B. At temperatures of 20 degrees C or lower, little or no inactivation of the enzyme was detected after 2 h of reaction with 50-300 microM concentrations of the reagent. However, incubation at 25 degrees C or higher resulted in an increased rate and extent of loss of the enzyme activity with increasing incubation temperatures. The Arrhenius plot for the inactivation process showed a sharp break at approximately 30 degrees C, with a heat of activation (delta H*) above this temperature of 55.2 kcal, indicating that a triethyltin-induced conformational change occurred at the elevated temperatures. Sugar substrates provided protection against the inactivating effect by reducing the binding of triethyltin to the enzyme. In the absence of glucose, two sites of different affinity for triethyltin exist in the hexokinase monomer. Binding of triethyltin to the enzyme shifted its monomer-dimer equilibrium toward the monomeric form in an early stage of the interaction. Inactivation of the enzyme was associated with a slower subsequent event. Comparative effects of various organotin compounds on the activity of the enzyme indicated that inhibitory potency was associated with increasing hydrophobicity of the alkyl groups attached to the tin.

Adenosine Triphosphatases↗
PubMed1983

Inactivation of yeast hexokinase B by triethyltin bromide and reactivation by dithiothreitol and glucose.

Binding of triethyltin bromide to yeast hexokinase B results in a rapid change in the reactivity of the sulfhydryl groups of the molecule. The change was characterized by an increased rate as well as extent of reaction of the -SH groups, and it preceded the onset of inhibition of the enzyme. Rapid gel filtration of the enzyme-triethyltin complex reversed this change in sulfhydryl reactivity, and when the eluted enzyme was subjected to short incubation periods, the slow inhibition that occurs with the unfiltered enzyme-triethyltin complex was no longer manifested. With prolonged incubation, however, the gel-filtered sample demonstrated increased rate of loss of enzyme activity, indicating that the gel filtration step did not completely reverse the effects of triethyltin on the enzyme. Active enzyme was recovered, following the inactivation of yeast hexokinase with triethyltin, by incubation of the inactivated enzyme with a large excess of glucose and dithiothreitol. Near total recovery of enzyme activity with reversion to native enzyme conformation was achieved following incubation at 35 degrees C of the enzyme with glucose and dithiothreitol each at 0.1 M. The possible involvement of either cysteine or histidine in the binding of triethyltin to the enzyme was probed, and it was concluded that neither of these amino acids are donor ligands for tin.

Dithiothreitol↗
PubMed1981

Thermodynamics of the binding of D-glucose to yeast hexokinase.

The binding of D-glucose to baker's yeast hexokinase (EC 2.7.1.1, ATP:D-hexose 6-phosphotransferase) was studied by isothermal and differential scanning calorimetry (DSC) and by fluorometric titration. The enthalpy and heat capacity changes associated with the binding of glucose were found to be nearly zero at both low and high ionic strengths over the temperature range from 7 to 29 degrees C. Thus, the free-energy change, amounting to -5.1 kcal mol(-1) at 25 degrees C and high ionic strength, is nearly independent of the temperature and is primarily of entropic origin. DSC study of the thermal unfolding of the free enzyme at low ionic strength gave an excess heat capacity curve with two maxima. This result appears to reflect a difference in thermal stability of the two domains in the hexokinase molecule which are indicated by X-ray crystallography [Bennett, W.S., & Steitz, T. A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852]. In contrast, the unfolding of free enzyme at high ionic strength was fully cooperative. The excess heat capacity curve for the unfolding of the glucose-bound enzyme had only one peak at both low and high ionic strengths. This is consistent with the X-ray result that the binding of glucose induces a conformational change in the enzyme which brings the two lobes into close proximity. It is interesting that such a significant, molecule-wide conformational change is accompanied by only very small net changes in enthalpy and heat capacity.

Binding Sites↗
PubMed1978

Fluorescence-quenching study of glucose binding by yeast hexokinase isoenzymes.

A study of the effect of varying ionic strength on the glucose-induced quenching of tryptophan fluorescence of hexokinase isoenzymes A(P-I) and B(P-II) was carried out at pH 8.3 and pH 5.5. At p/ 8.3 both isoenzymes gave apparently linear Scatchard-type data plots even with protein concentrations and ionic strengths for which both dimeric and monomeric forms of hexokinase coexist in signiciant amounts. Taking inco account a 1% accuracy in the experimental measurements, we concluded that the intrinsic dissociation constants K(M) and K(D), for the binding of glucose to the monomeric and dimeric forms of HkB, are within a factor of two of each other, i.e. K(D)/K(M) less than or equal to 2. The values of K(M), estimated from the apparent K, were so greatly influenced by ionic strength that it is clear that it is meaningless to compare K(M) and K(D) values measured at different ionic strengths as has been done in the literature. Curvature in the pH 5.5. fluorescence-quenching plots for relatively low ionic strengths demonstrates cooperativity for glucose-binding to the dimer, positive for HkA but negative for HkB. In contrast, the binding is relatively non-cooperative at high ionic strength at this pH. These results were attributed to the well known effect of salt-neutralization of side chain electrical charges on the flexibility and compactness of proteins.

Glucose↗