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PubMed2006

Adsorption behavior and activity of hexokinase.

The study on the adsorption of hexokinase (HK) onto silicon wafers was carried out by means of in situ ellipsometry and atomic force microscopy in the liquid. The thickness values of the adsorbed HK layer determined by both techniques were in excellent agreement and evidenced HK monolayer formation. The adsorption of HK onto Si wafers was favored at low ionic strength, indicating that the adsorption is mainly driven by electrostatic forces, since salt screens not only the segment-segment repulsion but also the segment-surface attraction when the salt concentration increases. The enzymatic activity of free HK and of adsorbed HK was measured as a function of time. Free HK in solution lost activity upon storage. Contrarily, adsorbed HK kept its activity level even after 48 h storage at room temperature. This outstanding behavior was attributed to specific orientation of the HK active site to the solution.

Adsorption↗
PubMed2005

Structural and functional implications of the hexokinase-nickel interaction.

The interaction between nickel and yeast hexokinase was studied. The binding of nickel showed a positive cooperativity, and saturation was not reached. The nickel binding induced modifications in the secondary structure of the protein; thus, a lost of alpha helix and beta turns, as well as an increase of the random structure and beta sheet was observed. The monomer/dimmer equilibrium of the protein was modified in the presence of nickel, and the monomer state was mainly obtained at the highest nickel concentrations studied. These changes on the protein structure caused a decrease in the enzyme activity. According to kinetic studies, nickel caused a non-competitive inhibition when glucose was the variable substrate and a linear competitive inhibition when ATP was the variable substrate.

Adenosine Triphosphate↗
PubMed2006

Effect of subchronic exposure to malathion on glycogen phosphorylase and hexokinase activities in rat liver using native PAGE.

The aim of this study was the evaluation of the effects of a subchronic exposure to malathion, an organophosphorus (OP) insecticide, on plasma glucose and hepatic enzymes of glycogenolysis and glycolysis in rats in vivo. Malathion was administered intragastrically by stomach tube in the amount of 1 ml corn oil containing 100mg/kg body weight (BW) daily for 32 days. At the end of the experiment, the liver was removed. The activities of glycogen phosphorylase (GP) and hexokinase (HK) were analysed in the homogenate. The methodology employed was a non-denaturing electrophoresis followed by activity-staining (native PAGE). Malathion decrease GP activity by 50% and increase HK activity by 10%. In addition, an hepatomegaly was recorded with a rise in the hepatic glycogen rate in malathion-treated rats. Moreover, subchronic administration of malathion has no effect on blood glucose concentration. The storage of glycogen in liver may be due to a stimulation of insulin secretion after the inhibition of acethylcholinesterase activity in pancreatic beta cells by malathion. These findings were in favour of an activation of glycogen storage by malathion.

Administration, Oral↗
PubMed2002

Mitochondrial bound type II hexokinase: a key player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention.

Despite more than 75 years of research by some of the greatest scientists in the world to conquer cancer, the clear winner is still cancer. This is reflected particularly by liver cancer that worldwide ranks fourth in terms of mortality with survival rates of no more than 3-5%. Significantly, one of the earliest discovered hallmarks of cancer had its roots in Bioenergetics as many tumors were found in the 1920s to exhibit a high glycolytic phenotype. Although research directed at unraveling the underlying basis and significance of this phenotype comprised the focus of cancer research for almost 50 years, these efforts declined greatly from 1970 to 1990 as research into the molecular and cell biology of this disease gained center stage. Certainly, this change was necessary as the new knowledge obtained about oncogenes, gene regulation, and programmed cell death once again placed Bioenergetics in the limelight of cancer research. Thus, we now have a much better molecular understanding of the high glycolytic phenotype of many cancers, the pivotal roles that Type II hexokinase-mitochondrial interactions play in this process to promote tumor cell growth and survival, and how this new knowledge can lead to improved therapies that may ultimately turn the tide on our losing war on cancer.

Cell Survival↗
PubMed1997

Source of ATP for hexokinase-catalyzed glucose phosphorylation in tumor cells: dependence on the rate of oxidative phosphorylation relative to that of extramitochondrial ATP generation.

We isolated highly intact and tightly coupled mitochondria from the rat ascites hepatoma cell line AH130 by disruption of the cell membrane by nitrogen cavitation. These isolated mitochondria were found to have essentially the same functional properties as rat liver mitochondria, but unlike the latter, hexokinase (HK) was bound to their membrane. Using the tumor mitochondrial preparation, we examined the source of ATP for phosphorylation of glucose by HK under conditions in which intra- and extramitochondrial ATP-generation systems operated separately or together. Results showed that the membrane-bound HK utilized ATP derived from the most efficiently operating ATP generation system, i.e., oxidative phosphorylation. However, when the rate of extramitochondrial ATP generation was much greater than that of oxidative phosphorylation, HK used ATP from the extramitochondrial ATP-generation system.

Adenosine Diphosphate↗
PubMed1998

Quantitative determinations of the steady state transcript levels of hexokinase isozymes and glucose transporter isoforms in normal rat tissues and the malignant tumor cell line AH130.

The steady state transcript levels of the four hexokinase (HK) isozymes and four glucose transporter (GLUT) isoforms were determined quantitatively by Northern analysis of RNA samples from rat tissues using synthetic fragments of the RNAs encoding the HK isozymes and GLUT isoforms. Results showed that the levels of HK isozyme transcripts were low in rat tissues, the level of that most highly expressed, the type I isozyme (HKI), in the brain being 0.025% of the total poly(A)+ RNA. A good correlation was found between the reported HK activities and the total amounts of transcripts encoding all HK isozymes in various tissues, showing that the HK activities in tissues can be estimated from the total amount of transcripts encoding HK isozymes. The proposed associated expressions of HK isozymes and GLUT isoforms in particular tissues were confirmed at their transcript levels. The steady state transcript levels of type II HK and the type 1 GLUT isoform in the malignant tumor cell line AH130 were also determined quantitatively.

Animals↗
PubMed2002

Mediator factor Med8p interacts with the hexokinase 2: implication in the glucose signalling pathway of Saccharomyces cerevisiae.

In the presence of glucose the protein hexokinase 2 (Hxk2p), normally resident in the cytosol, is translocated to the nucleus where it impairs the activation of transcription of the glucose-repressed genes HXK1, GLK1 and SUC2, and promotes the activation of transcription of the glucose-induced genes HXK2 and HXT1. Here, we demonstrate the involvement of an heptameric motif, named the MED8 site, in the direct binding of the mediator protein Med8p, either as a monomer or as a homodimer. Because this site was previously involved in the Hxk2p-dependent glucose-induced regulation of gene transcription, we tested whether Hxk2p interacts with Med8p. Our results show that Hxk2 and Med8 proteins are physically associated and that this Hxk2p-Med8p interaction is of physiological significance because both proteins have been found interacting together in a cluster with DNA fragments containing the MED8 site. We conclude that Hxk2p operates through the MED8 site, by interacting with Med8p, in the glucose signal transduction pathway of Saccharomyces cerevisiae.

DNA-Binding Proteins↗
PubMed1996

Quantitative enzyme- and immunohistochemistry of hexokinase and cytochrome c oxidase of spinal neurons in the zebrafish.

A combined quantitative enzyme- and immunohistochemical procedure to demonstrate hexokinase (HK) was developed and tested on sections of spinal cord tissue of the zebrafish. In both procedures, the amount of final reaction product was linearly related with section thickness. When applied to serial sections of fish spinal neurons, the enzyme- and immunohistochemical activities appeared to correlate significantly (r = 0.61; p < 0.001). As HK and cytochrome c oxidase (COX) histochemistry have been used regularly to screen the average level of chronic activity of neurons, we subsequently analysed the relationship between HK and COX in fish spinal neurons, using previously published methods of quantitative enzyme- and immunohistochemistry for COX. The enzyme- as well as the immunohistochemical localisation patterns of HK showed a weak correlation with the enzyme- and immunohistochemical COX localisation respectively. Therefore, it is concluded that both enzyme- and immunohistochemical localisation of COX provide a poor estimate for the relative level of glucose utilisation in fish spinal neurons.

Adenosine Triphosphate↗
PubMedDate not supplied

Hexokinase isoenzymes in the rat placenta.

The phosphorylation of glucose to glucose-6-phosphate, the first enzymatic step for glucose utilization is catalysed by a family of four hexokinase isoenzymes (HKI-IV) which display a tissue-specific distribution. The expression of HK isoenzymes was investigated in the rat placenta. High levels of HKI and HKII mRNA were found in the junctional and the labyrinthine zones. HKIII mRNA was present at low levels in the junctional zone and glucokinase (HKIV) mRNA was not detected, indicating that HKI and HKII are the two major placental HK isoenzymes. HKII activity was increased in placenta of insulinopenic diabetic rats. This regulation is likely to support the increase in glucose utilization and storage characteristics of the enlarged placentae of diabetic rats.

Animals↗
PubMed2002

Studies of cadmium binding to hexokinase: structural and functional implications.

The interaction between cadmium and yeast hexokinase was studied. Cadmium produces changes in the aggregation state of the protein and large structures with a large molecular mass were formed. This phenomenon occurs without large modifications to the secondary structure. During this change the enzyme maintains a high level of activity in the monomer as well as in aggregate form. This implies that the enzyme function is not greatly affected by the change and it maintains its active sites without significant modifications. According to kinetic measurements with both glucose and ATP as a variable substrate, cadmium causes a mixed-type inhibition with a main uncompetitive component. Binding experiments show that the protein presents negative cooperative binding with cadmium at various temperatures (298, 303 and 313 K) and a progressive loss in metal union with concentration depending on the temperature. The total union percentage decreases as the metal concentration increases. This is probably due to the aggregation process, which affects the binding sites for the metal and also for the substrate. Labile interactions are more persistent than specific interactions in accordance with the solvation parameter.

Cadmium↗
PubMed2000

Structure of the 5' region of the human hexokinase type I (HKI) gene and identification of an additional testis-specific HKI mRNA.

We previously reported the structure of the human hexokinase type I (HKI) gene and provided direct evidence of an alternative red blood cell-specific exon 1 located in the 5' flanking region of the gene. Three unique HKI mRNA species have also been described in human spermatogenic cells. These mRNAs contain a testis-specific sequence not present in somatic cell HKI, but lack the sequence for the porin-binding domain necessary for HKI to bind to porin on the outer mitochondrial membrane. The present study reports a new mRNA isoform, hHKI-td, isolated from human sperm. hHKI-td mRNA contains both a testis-specific sequence at the 5' end common to the three other mRNA isoforms and an additional unique sequence. Screening of a cosmid library and analysis of the cosmids containing the HKI gene revealed that testis-specific sequences are encoded by six different exons. Five of these exons are located upstream from the somatic exon 1 (5.6-30 kb) and one within intron 1. This study shows that a single human HKI gene spanning at least 100 kb encodes multiple transcripts that are generated by alternative splicing of different 5' exons. Testis-specific transcripts are probably produced by a separate promoter that induces the expression of the HKI gene in spermatogenic cells.

Amino Acid Sequence↗
PubMed2002

Effects of phosphate deficiency and sugars on expression of rab18 in Arabidopsis: hexokinase-dependent and okadaic acid-sensitive transduction of the sugar signal.

The lack of phosphorus in the nutrient medium increased the expression of rab18, an abscisic acid (ABA)-responsive gene, in leaves of Arabidopsis thaliana. The expression of this gene was also upregulated after feeding the excised leaves with D-mannose and sucrose for both wild-type (wt) and aba1 (ABA-deficient) mutant plants. For aba1 mutants, both the phosphate deficiency and sugar effects on rab18 were weaker than in wt plants, suggesting possible involvement of both ABA-dependent and ABA-independent components in signalling. Transgenic Arabidopsis plants with increased hexokinase (HXK) expression had a much higher sucrose-dependent level of rab18 mRNA, implying the HXK involvement in sensing/transmitting the sugar signal. Sucrose-related induction of rab18 was completely inhibited by okadaic acid (OKA), suggesting the involvement of specific protein phosphatase(s) in transduction of the sugar signal. The results suggest that rab18 is regulated via interaction of a plethora of signals, including ABA, sugar and phosphate deficiency, and that the sugar effect is transmitted via a HXK-pathway, involving OKA-sensitive component(s). The findings prompt caution in linking the expression of rab18 solely to ABA signalling.

Abscisic Acid↗
PubMed2001

Growth condition-dependent synchronized changes in transcript levels of type II hexokinase and type 1 glucose transporter in tumor cells.

Transcript levels of hexokinase (HK) isozymes and glucose transporter (GLUT) isoforms in RNA samples of AH130 cells obtained from dish cultures and ascites were evaluated in a quantitative manner. In AH130 cells cultured in dishes, HKI and HKII were expressed at a similar level, but HKIII and HKIV were not. GLUT1 and GLUT3 were also expressed, and messages of these two isoforms represented 27% and 71%, respectively, of the total GLUT messages. A faint signal of GLUT2 was also observed. On the contrary, in cells grown as ascites, the transcript of HKII was dominant, and its level was about 15-fold over that of dish-cultured AH130 cells. Transcript levels of GLUT1 and GLUT3 were 4.5- and 2-fold, respectively, higher than those in dish-cultured cells. Thus, GLUT1 was more susceptible to changes in culture conditions than GLUT3. Based on these results, we concluded that the change in growth conditions caused synchronized changes in the transcript levels of HKII and GLUT1 in AH130 cells. However, such marked changes in the transcript levels of HKII and GLUT1 were not observed when AH130 cells were cultured in dishes under a hypoxic condition, indicating that the observed changes were not solely attributable to the difference in oxygen concentration between the ascites and cell culture conditions. Accordingly, other factors such as growth factors may be responsible for this difference in levels of HKII and GLUT1 between the two growth conditions.

Animals↗
PubMed2004

Hexokinase II and VEGF expression in liver tumors: correlation with hypoxia-inducible factor 1 alpha and its significance.

BACKGROUND/AIMS: We analyzed the expressions of hexokinase II (HK II), a key enzyme in glycolysis, and VEGF in hepatocellular carcinoma (HCC) and metastatic liver cancer in relation to tumor vascularity, and the participation of hypoxia-inducible factor-1 (HIF-1) was studied. METHODS: A real-time quantitative reverse transcription-polymerase chain reaction was performed to examine the HK II and VEGF mRNA expression. Expression of HIF-1 alpha and HK II protein, and microvessel density (MVD) were examined immunohistochemically. RESULTS: MVD was significantly higher in HCCs than in metastatic liver cancers, and VEGF mRNA expression was positively correlated only with MVD of HCCs. HK II mRNA expression was significantly higher in metastatic liver cancers, however, some cases of HCC pretreated with transcatheter arterial embolization (TAE) showed marked HK II mRNA expression. Both HIF-1 alpha and HK II protein expressions were co-localized in the cancer cells near necrosis, and the intensity of HIF-1 alpha protein expression was significantly correlated with HK II mRNA expression in both tumors. CONCLUSIONS: These results suggest that, in metastatic liver cancers, glycolysis induced by HIF-1 is the predominant energy source under the hypoxic environment and, at least in some TAE-pretreated HCC cases, cancer cells obtain energy for growth by switching the metabolic profile to glycolysis through HIF-1.

Aged↗
PubMed2001

Influence of the oligomeric state of yeast hexokinase isozymes on inactivation and unfolding by urea.

The effect of the association-dissociation equilibrium on the urea-induced inactivation and unfolding of the yeast hexokinase isoforms, PI and PII, showed that these enzymes are more stable as dimers. For the monomeric PII, the inactivation and unfolding processes occurred in parallel. However, inactivation precedes the unfolding of monomeric PI or dimeric PI and PII. The unfolding transitions are biphasic for PI indicating stable intermediates, whereas for the PII isoform the unfolding occurs in a single step. Our data suggests that although PI and PII present a 78% identity in their amino acid sequences, they probably have distinct inactivation and unfolding by urea behavior.

Biopolymers↗
PubMed2003

Leporin B: a novel hexokinase II gene inducing agent from an unidentified fungus.

Leporin B (1), a novel demethylated analogue of leporin A (2), was isolated from a taxonomically unidentified fungal strain as part of an effort to discover compounds with the ability to increase expression levels of the enzyme hexokinase II. The structure was determined by spectral methods, including 1D and 2D NMR, and HRMS. The relative stereochemistry was assigned by NOESY experiments and coupling constants.

Animals↗
PubMed2001

Kinetic characterization of hexokinase isoenzymes from glioma cells: implications for FDG imaging of human brain tumors.

Quantitative imaging of glucose metabolism of human brain tumors with PET utilizes 2-[(18)F]-fluorodeoxy-D-glucose (FDG) and a conversion factor called the lumped constant (LC), which relates the metabolic rate of FDG to glucose. Since tumors have greater uptake of FDG than would be predicted by the metabolism of native glucose, the characteristic of tumors that governs the uptake of FDG must be part of the LC. The LC is chiefly determined by the phosphorylation ratio (PR), which is comprised of the kinetic parameters (Km and Vmax) of hexokinase (HK) for glucose as well as for FDG (LC proportional to (Km(glc) x Vmax(FDG))/(Km(FDG) x Vmax(glc)). The value of the LC has been estimated from imaging studies, but not validated in vitro from HK kinetic parameters. In this study we measured the kinetic constants of bovine and 36B-10 rat glioma HK I (predominant in normal brain) and 36B-10 glioma HK II (increased in brain tumors) for the hexose substrates glucose, 2-deoxy-D-glucose (2DG) and FDG. Our principal results show that the KmGlc < KmFDG << Km2DG and that PR2DG < PRFDG. The FDG LC calculated from our kinetic parameters for normal brain, possessing predominantly HK I, would be higher than the normal brain LC predicted from animal studies using 2DG or human PET studies using FDG or 2DG. These results also suggest that a shift from HK I to HK II, which has been observed to increase in brain tumors, would have little effect on the value of the tumor LC.

Animals↗
PubMed2002

Effect of dye aggregation on triarylmethane-mediated photoinduced damage of hexokinase and DNA.

The observation that enhanced mitochondrial membrane potential is a prevalent cancer cell phenotype has provided the conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for both chemo- and photochemotherapy of neoplastic diseases. Cationic triarylmethane (TAM(+)) dyes represent a series of photosensitizers whose phototoxic effects develop at least in part at the mitochondrial level. In this report we describe how the molecular structure of four representative TAM(+) dyes (Crystal Violet, Ethyl Violet, Victoria blue R, and Victoria pure blue BO) affects their efficiency as mediators of the photoinduced inactivation of two model mitochondrial targets, hexokinase (HK) and DNA. Our results have indicated that TAM(+) dyes efficiently bind to HK and DNA in aqueous media both as dye monomers and aggregates, with the degree of aggregation increasing with increasing the lipophilic character of the photosensitizer. The efficiency with which HK and DNA are damaged upon 532 nm photolysis of biopolymer-TAM(+) complexes was found to decrease upon increasing the degree of dye aggregation over these macromolecular templates. Comparative experiments carried out both in water and in D(2)O, and in air-equilibrated and nitrogen-purged samples have also indicated that, at least when Crystal Violet is used as the photosensitizer, the mechanism of macromolecular damage does not require the involvement of molecular oxygen to operate. This finding makes Crystal Violet a potential candidate for use in photochemotherapy of hypoxic or poorly perfused tumor areas.

Coloring Agents↗