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PubMed1994

The soluble but not mitochondrially bound hexokinase is a substrate for the ATP- and ubiquitin-dependent proteolytic system.

Intracellular protein degradation is highly selective, however, the mechanism(s) underlying this selectivity are not fully understood. We have previously shown that purified rabbit hexokinase type I, an enzyme present in mammalian brain both in soluble and mitochondrial bound form, is conjugate to ubiquitin and then degraded by a rabbit reticulocyte fraction II. In the present study we report that the mitochondrial bound hexokinase is stable for several hours in the same proteolytic system both in the presence or absence of ATP. E1, E2 and E3, the enzymes of the ubiquitin conjugating system, are able to incorporate 125I- or biotin-labelled ubiquitin in an ATP-dependent manner in soluble hexokinase as well as in a number of mitochondrial proteins. Furthermore, the mitochondria by themselves have a pronounced ATP-dependent ability to conjugate 125I-ubiquitin. However, Western blotting experiments, using a specific antibody against hexokinase, or against ubiquitin, showed that the mitochondrial bound enzyme is neither ubiquitinated nor degraded. This result has been confirmed by purification of bound hexokinase from the brain mitochondrial fraction or following the incubation of intact mitochondria with ATP, 125I-ubiquitin and E1, E2 and E3. Thus, mitochondrial bound hexokinase is not recognized by the ubiquitin conjugating system while the soluble enzyme is conjugate to ubiquitin and then degraded. Furthermore, the soluble hexokinase from rabbit brain was isolated by immunoaffinity chromatography and shown to be recognized by an anti-ubiquitin antibody. These results suggest that the intracellular distribution of protein is an important feature of a protein which determines its susceptibility to ubiquitin-dependent degradation.

Adenosine Triphosphate↗
PubMed1988

Improved metabolic properties of hexokinase-overloaded human erythrocytes.

Human erythrocytes were loaded with homogeneous hexokinase purified from human placenta (an enzyme species apparently identical to the erythrocyte enzyme), using a procedure of encapsulation based on hypotonic hemolysis, isotonic resealing and reannealing. The hexokinase-overloaded erythrocytes contained 4.77 +/- 0.75 IU of hexokinase activity per ml of packed erythrocytes, a value 15-times higher than that of corresponding unloaded or native red cells. The hexokinase-loaded erythrocytes were found to metabolize twice the amount of glucose consumed by the unloaded cells through a nearly doubled glycolytic activity, while the activity of the hexose monophosphate shunt pathway was unmodified. Estimates of glycolytic intermediates showed increased levels of most metabolites with respect to the unloaded erythrocytes, while the intracellular concentrations of adenine nucleotides and 2,3-bisphosphoglycerate were unaffected by entrapment of hexokinase. The new steady-state condition characterized by improved glycolytic function was demonstrated to be directly related to enhanced levels of hexokinase activity and not to the use of a rejuvenation solution during the procedure of entrapment. These results are consistent with suggestions by several investigators that glucose metabolism in human erythrocytes is regulated by hexokinase, and they open new perspectives for manipulating erythrocytes with the ultimate aim of improving their survival under different storage conditions.

Adenine Nucleotides↗
PubMed1983

Polyamines stimulate the binding of hexokinase type II to mitochondria.

Spermine and spermidine enhanced the binding of hexokinase isoenzyme type II to mitochondria, both of which were prepared from Ehrlich-Lettre hyperdiploid ascites tumor cells, at much lower concentrations than Mg2+. Chymotrypsin-treated hexokinase II could not bind to the mitochondrial membrane in the presence of either spermine or Mg2+, indicating that the effect of spermine is not a nonspecific action, since the treatment of chymotrypsin cleaves only the region essential for the binding without any significant effect of the catalytic activity. Both spermine and Mg2+ antagonized the glucose 6-phosphate-induced release of mitochondria-bound hexokinase, and promoted the binding of the solubilized hexokinase II even in the presence of glucose 6-phosphate. However, inhibition of the activity of soluble hexokinase by glucose 6-phosphate was not reversed by spermine and Mg2+. Hexokinase II rebound to mitochondria with spermine and Mg2+ produced glucose 6-phosphate using ATP generated inside the mitochondria, and no difference was observed between the spermine- and Mg2+-rebound systems. Significance of the binding of hexokinase to mitochondria, especially with polyamines, is discussed with reference to high glycolytic rate in tumor cells.

Animals↗
PubMed1985

Hexokinase in human chorionic villi.

The level, intracellular distribution, and isozymic pattern of hexokinase (EC 2.7.1.1) were determined on human chorionic villi obtained by trophoblast biopsy in the first trimester of pregnancy. About 50% of total hexokinase activity was found to be particle-bound and 96% of this in the overt form (i.e. assayable without the addition of detergents). Both soluble and particulate hexokinase show the same affinities for glucose but differ in the affinity for MgATP2- and in their sensitivity to glucose 1,6-diphosphate inhibition. By chromatographic and kinetic methods the soluble hexokinase was found to be represented by isozymes I, II and traces of hexokinase III while most of the bound enzyme was hexokinase type I. These results provide evidence for the expression of at least three hexokinase isozymes in early human development and can partially explain the high rate of glucose utilization of the placenta.

Adenosine Triphosphate↗
PubMed2005

Glucose inhibits the expression of triose phosphate/phosphate translocator gene in wheat via hexokinase-dependent mechanism.

Although triose phosphate/phosphate translocator is known to play an important role in regulating the distribution of assimilates in wheat chloroplasts, the mechanism of triose phosphate/phosphate translocator gene control has not yet been clearly elucidated. We first showed that glucose inhibited the expression of triose phosphate/phosphate translocator gene in wheat by reverse transcription-polymerase chain reaction and Western blotting. The triose phosphate/phosphate translocator expression was seriously impaired by 5 mmol/L glucose, and it responded slowly, more than 48 h, to level as low as 1 mmol/L glucose. Both glucose and 2-deoxyglucose inhibited the expression of triose phosphate/phosphate translocator gene, but 2-deoxyglucose-6-P, product of phosphorylated 2-deoxyglucose, cannot be further metabolized, therefore the further metabolism of phosphorylated glucose by hexokinase is not a prerequisite for triggering glucose-regulated expression of triose phosphate/phosphate translocator gene. Glucose had little inhibitory effect on the expression of triose phosphate/phosphate translocator gene when hexokinase activity was reduced or eliminated by transforming wheat protoplasts with a hexokinase antisense construct or treating protoplasts with glucosamine, an inhibitor of hexokinase. Therefore, it appears essential for hexokinase to retain phosphorylation activity for glucose to regulate the expression of triose phosphate/phosphate translocator gene. The treatment of protoplasts with glucose-6-phosphate resulting in no inhibition of triose phosphate/phosphate translocator expression demonstrated that phosphorylation via hexokinase is necessary for glucose inhibiting triose phosphate/phosphate translocator expression. All the data suggest that triose phosphate/phosphate translocator is regulated by glucose via a hexokinase-dependent pathway.

Chloroplast Proteins↗
PubMed1997

Hexokinase II activity and messenger ribonucleic acid abundance in adipose tissue and skeletal muscle during pregnancy and lactation in rats.

OBJECTIVE: Our purpose was to investigate the influence of pregnancy and lactation on hexokinase II activity and gene expression in adipose tissue and skeletal muscle of rats. STUDY DESIGN: Intraabdominal adipose tissue and femoral quadriceps muscle were removed from Sprague-Dawley rats at various times (6 to 10 animals per group) during pregnancy and lactation. Hexokinase II messenger ribonucleic acid was assayed by quantitative ribonuclease protection analysis, and hexokinase activity was measured with the glucose-6-phosphate-coupled assay. RESULTS: The amount of hexokinase II messenger ribonucleic acid in adipose tissue on day 20 of pregnancy was 55.4% +/- 8.4% (p < 0.01, unpaired Student t test) of the value for nonpregnant control rats; hexokinase II activity was also decreased in this tissue at this time (39.34 +/- 3.05 vs 27.81 +/- 3.61 mU x min(-1) x mg protein(-1), p < 0.05). Hexokinase II activity and messenger ribonucleic acid abundance in skeletal muscle were unaffected by pregnancy and lactation. CONCLUSION: Both hexokinase II messenger ribonucleic acid abundance and enzyme activity are reduced in adipose tissue of pregnant rats near term, possibly contributing to the insulin resistance associated with late pregnancy.

Adipose Tissue↗
PubMed2001

Regulation of phosphotransferase activity of hexokinase 2 from Saccharomyces cerevisiae by modification at serine-14.

Isoenzyme 2 of hexokinase functions in sugar sensing and glucose repression in Saccharomyces cerevisiae. The degree of in vivo phosphorylation of hexokinase 2 at serine-14 is inversely related to the extracellular glucose concentration [Vojtek, A. B., and Fraenkel, D. G. (1990) Eur. J. Biochem. 190, 371-375]; however, a physiological role of the modification causing the dissociation of the dimeric enzyme in vitro [as effected by a serine-glutamate exchange at position 14; Behlke et al. (1998) Biochemistry 37, 11989-11995] is unclear. This paper describes a comparative stopped-flow kinetic and sedimentation equilibrium analysis performed with native unphosphorylated hexokinase 2 and a permanently pseudophosphorylated glutamate-14 mutant enzyme to determine the functional consequences of phosphorylation-induced enzyme dissociation. The use of a dye-linked hexokinase assay monitoring proton generation allowed the investigation of the kinetics of glucose phosphorylation over a wide range of enzyme concentrations. The kinetic data indicated that monomeric hexokinase represents the high-affinity form of isoenzyme 2 for both glycolytic substrates. Inhibition of glucose phosphorylation by ATP [Moreno et al. (1986) Eur. J. Biochem. 161, 565-569] was only observed at a low enzyme concentration, whereas no inhibition was detected at the high concentration of hexokinase 2 presumed to occur in the cell. Pseudophosphorylation by glutamate substitution for serine-14 increased substrate affinity at high enzyme concentration and stimulated the autophosphorylation of isoenzyme 2. The possible role of hexokinase 2 in vivo phosphorylation at serine-14 in glucose signaling is discussed.

Amino Acid Substitution↗
PubMed1968

The effect of anti-insulin serum and alloxan-diabetes on the distribution and multiple forms of hexokinase in lactating rat mammary gland.

1. The distribution and multiple forms of hexokinase activity in lactating rat mammary gland were investigated in alloxan-diabetic rats and in rats treated with anti-insulin serum. It was found that 46% of the total hexokinase of mammary-gland tissue from control rats was in the particulate fraction, but this percentage was decreased in the alloxan-diabetic rats to 11% of the total hexokinase. The hexokinase activity of the soluble fraction was not significantly altered but there was a decrease in the type II/type I quotient. 2. The early changes that occurred on insulin deprivation were studied 1hr. after administration of anti-insulin serum to lactating rats, at which time the hexokinase bound to the particulate fraction had decreased to 11% of the control value and that in the soluble fraction had increased by approx. 50%. The hexokinase type II/type I quotient in the soluble fraction was significantly decreased. These results suggested that there was a release of particulate-bound hexokinase in rats treated with anti-insulin serum.

Animals↗
PubMed1994

Hexokinase and glucokinase binding in permeabilized guinea-pig hepatocytes.

The release of glucokinase (hexokinase IV) from digitonin-permeabilized hepatocytes from rat, guinea pig or mouse liver is inhibited by physiological concentrations of Mg2+ (> 0.25 mM). Preincubation of hepatocytes with fructose increases glucokinase release during permeabilization in the presence of Mg2+ but decreases glucokinase release in the absence of Mg2+, suggesting that fructose causes translocation of glucokinase from the Mg(2+)-dependent site. Glucose (25 mM) and sorbitol (1 mM) also induce translocation of glucokinase from the Mg(2+)-dependent site in guinea-pig, as in rat hepatocytes, but glucose is less effective than fructose or sorbitol, and the concentrations of fructose and sorbitol that cause half-maximal activation (A50) are 3-fold and 20-fold higher, respectively, in guinea-pig than in rat hepatocytes (170 microM and 257 microM, compared with 61 microM and 13 microM). Dihydroxyacetone and glycerol have no effect on fructose-induced or sorbitol-induced translocation in guinea-pig hepatocytes, in contrast with the potentiation and inhibition, respectively, by these substrates in rat hepatocytes. Some, but not all, of the differences between rat and guinea-pig hepatocytes could be due to the more reduced cytoplasmic NADH/NAD+ redox state in guinea-pig cells. The activity of low-Km hexokinases accounts for 30% of total hexokinase activity (low-Km hexokinases + glucokinase) in guinea-pig hepatocytes. Of the low-Km hexokinase activity, approx. 30% is released in the presence of Mg2+, 9% shows Mg(2+)-dependent binding and 60% shows Mg(2+)-independent binding. There was no substrate-induced translocation of low-Km hexokinase activity, indicating that translocation is specific for hexokinase IV.

Animals↗
PubMed2000

Binding of rat brain hexokinase to recombinant yeast mitochondria: identification of necessary physico-chemical determinants.

The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harboring human porin (Yh) is comparable to that with rat liver mitochondria in terms of cation requirements, cooperativity in binding, and the effect of amphipathic compounds. Mg2+, which is required for hexokinase binding to all mitochondria, can be replaced by other cations. The efficiency of hexokinases, however, depends on the valence of hydrophilic cations, or the partition of hydrophobic cations in the membrane, implying that these act by reducing a prohibitive negative surface charge density on the outer membrane rather than fulfilling a specific structural requirement. Macromolecular crowding (using dextran) has dual effects. Dextran added in excess increases hexokinase binding to yeast mitochondria, according to the porin molecule they harbor. This effect, significant with wild-type yeast mitochondria, is only marginal with Yh as well as rat mitochondria. On the other hand, an increase in the number of hexokinase binding sites on mitochondria is also observed. This increase, moderate in wild-type organelles, is more pronounced with Yh. Finally, dextran, which has no effect on the modulation of hexokinase binding by cations, abolishes the inhibitory effect of amphipathic compounds. Thus, while hexokinase binding to mitochondria is predetermined by the porin molecule, the organization of the latter in the membrane plays a critical role as well, indicative that porin must associate with other mitochondrial components to form competent binding sites on the outer membrane.

Animals↗
PubMed1999

Hexokinase II-deficient mice. Prenatal death of homozygotes without disturbances in glucose tolerance in heterozygotes.

Type 2 diabetes is characterized by decreased rates of insulin-stimulated glucose uptake and utilization, reduced hexokinase II mRNA and enzyme production, and low basal levels of glucose 6-phosphate in insulin-sensitive skeletal muscle and adipose tissues. Hexokinase II is primarily expressed in muscle and adipose tissues where it catalyzes the phosphorylation of glucose to glucose 6-phosphate, a possible rate-limiting step for glucose disposal. To investigate the role of hexokinase II in insulin action and in glucose homeostasis as well as in mouse development, we generated a hexokinase II knock-out mouse. Mice homozygous for hexokinase II deficiency (HKII(-/-)) died at approximately 7.5 days post-fertilization, indicating that hexokinase II is vital for mouse embryogenesis after implantation and before organogenesis. HKII(+/-) mice were viable, fertile, and grew normally. Surprisingly, even though HKII(+/-) mice had significantly reduced (by 50%) hexokinase II mRNA and activity levels in skeletal muscle, heart, and adipose tissue, they did not exhibit impaired insulin action or glucose tolerance even when challenged with a high-fat diet.

Adipose Tissue↗
PubMed2003

A novel type of chloroplast stromal hexokinase is the major glucose-phosphorylating enzyme in the moss Physcomitrella patens.

Hexokinase catalyzes the first step in the metabolism of glucose but has also been proposed to be involved in sugar sensing and signaling both in yeast and in plants. We have cloned a hexokinase gene, PpHXK1, in the moss Physcomitrella patens where gene function can be studied directly by gene targeting. PpHxk1 is a novel type of chloroplast stromal hexokinase that differs from previously studied membrane-bound plant hexokinases. Enzyme assays on a knock-out mutant revealed that PpHxk1 is the major glucose-phosphorylating enzyme in Physcomitrella, accounting for 80% of the total activity in protonemal tissue. The mutant is deficient in the response to glucose, which in wild type moss induces the formation of caulonemal filaments that protrude from the edge of the colony. Growth on glucose in the dark is strongly reduced in the mutant. Sequence data suggest that most plants including Physcomitrella and Arabidopsis have both chloroplast-imported hexokinases similar to PpHxk1 and traditional membrane-bound hexokinases. We propose that the two types of plant hexokinases have distinct physiological roles.

Amino Acid Sequence↗
PubMed2006

Suppression of apoptosis by cyclophilin D via stabilization of hexokinase II mitochondrial binding in cancer cells.

The permeability transition pore is involved in the mitochondrial pathway of apoptosis. Cyclophilin D, a pore component, has catalytic activity as a peptidyl prolyl cis, trans-isomerase (PPIase), which is essential to the pore opening. It has been reported that cyclophilin D overexpression suppresses apoptosis in cancer cells. To clarify the mechanism of this effect, we generated glioma cells overexpressing wild-type or a PPIase-deficient mutant of cyclophilin D. Interestingly, we found that the PPIase-dependent apoptosis suppression by cyclophilin D correlated with the amounts of mitochondrial-bound hexokinase II, which has anti-apoptotic activity. Inactivation of endogenous cyclophilin D by small interference RNA or a cyclophilin inhibitor was found to release hexokinase II from mitochondria and to enhance Bax-mediated apoptosis. The anti-apoptotic effects of cyclophilin D were canceled out by the detachment of hexokinase II from mitochondria, demonstrating that mitochondrial binding of hexokinase II is essential to the apoptosis suppression by cyclophilin D. Furthermore, cyclophilin D dysfunction appears to abrogate hexokinase II-mediated apoptosis suppression, indicating that cyclophilin D is required for the anti-apoptotic activity of hexokinase II. Based on the above, we propose here that cyclophilin D suppresses apoptotic cell death via a mitochondrial hexokinase II-dependent mechanism in cancer cells.

Animals↗
PubMed2007

Crystal structures of an ATP-dependent hexokinase with broad substrate specificity from the hyperthermophilic archaeon Sulfolobus tokodaii.

Hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate by using ATP as a phosphoryl donor. Recently, we identified and characterized an ATP-dependent hexokinase (StHK) from the hyperthermophilic archaeon Sulfolobus tokodaii, which can phosphorylate a broad range of sugar substrates, including glucose, mannose, glucosamine, and N-acetylglucosamine. Here we present the crystal structures of StHK in four different forms: (i) apo-form, (ii) binary complex with glucose, (iii) binary complex with ADP, and (iv) quaternary complex with xylose, Mg(2+), and ADP. Forms i and iii are in the open state, and forms ii and iv are in the closed state, indicating that sugar binding induces a large conformational change, whereas ADP binding does not. The four different crystal structures of the same enzyme provide "snapshots" of the conformational changes during the catalytic cycle. StHK exhibits a core fold characteristic of the hexokinase family, but the structures of several loop regions responsible for substrate binding are significantly different from those of other known hexokinase family members. Structural comparison of StHK with human N-acetylglucosamine kinase and other hexokinases provides an explanation for the ability of StHK to phosphorylate both glucose and N-acetylglucosamine. A Mg(2+) ion and coordinating water molecules are well defined in the electron density of the quaternary complex structure. This structure represents the first direct visualization of the binding mode for magnesium to hexokinase and thus allows for a better understanding of the catalytic mechanism proposed for the entire hexokinase family.

Adenosine Triphosphate↗
PubMed2001

Changes in leaf hexokinase activity and metabolite levels in response to drying in the desiccation-tolerant species Sporobolus stapfianus and Xerophyta viscosa.

The phosphorylation of glucose and fructose is an important step in regulating the supply of hexose sugars for biosynthesis and metabolism. Changes in leaf hexokinase (EC 2.7.1.1) activity and in vivo metabolite levels were examined during drying in desiccation-tolerant Sporobolus stapfianus and Xerophyta viscosa. Leaf hexokinase activity was significantly induced from 85% to 29% relative water content (RWC) in S. stapfianus and from 89% to 55% RWC in X. viscosa. The increase in hexokinase corresponded to the region of sucrose accumulation in both species, with the highest activity levels coinciding with region of net glucose and fructose removal. The decline of hexose sugars and accumulation of sucrose in both plant species was not associated with a decline in acid and neutral invertase. The increase in hexokinase activity may be important to ensure that the phosphorylation and incorporation of glucose and fructose into metabolism exceeded production from potential hydrolytic activity. Total cellular glucose-6-phosphate (Glc-6-P) and fructose-6-phosphate (Fru-6-P) levels were held constant throughout dehydration. In contrast to hexokinase, fructokinase activity was unchanged during dehydration. Hexokinase activity was not fully induced in leaves of S. stapfianus dried detached from the plant, suggesting that the increase in hexokinase may be associated with the acquisition of desiccation-tolerance.

Carbohydrate Metabolism↗
PubMed1980

Increase in sperm type hexokinase activity of rat spermatozoa during maturation.

Rat hexokinases fro caput sperm (immature) and caudal sperm (mature) were investigated. The hexokinases from both sources were studied by DEAE-cellulose column chromatography and by cellulose acetate electrophoresis. The specific activity of caput sperm hexokinase was not significantly different from that of the caudal sperm enzyme. Spermatozoa possess two isozymes of hexokinase. Type I hexokinase was the predominant type in caput sperm whereas the sperm type of hexokinase was predominant in caudal sperm. Hexokinase types II and III were absent in both extracts.

Animals↗
PubMed1980

Localization of hexokinase in neural tissue: immunofluorescence studies on the developing cerebellum and retina of the rat.

Antiserum against purified rat brain hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) has been used in a study of the distribution of hexokinase during the postnatal development of rat cerebellum and retina. The cells of the external germinal layer of the cerebellum exhibit little or no fluorescence. The Purkinje cells exhibit a transient increase in hexokinase levels between 2 and 8 days postnatally, followed by a precipitous decrease (18--12 days) to the relatively low levels found in the mature Purkinje cell. Development of the intensely fluorescent cerebellar glomeruli in the granule cell layer is readily followed during the 3rd and 4th weeks postnatally. With respect to postnatal changes in hexokinase distribution of the retina, perhaps most notable is the observation that even the cytoplasmic protrusions which represent the precursors of the photoreceptor segments are richly endowed with hexokinase. Biochemical differentiation of the photoreceptor segments into hexokinase-rich inner segments and hexokinase-poor outer segments is readily observed long before the growth of the photoreceptor segments has been completed.

Aging↗
PubMed1981

Competitive inhibition of rat brain hexokinase by 2-deoxyglucose, glucosamine, and metrizamide.

The effects of metrizamide on the kinetics of rat brain hexokinase were compared in vitro with those of 2-deoxyglucose and glucosamine. Although metrizamide, 2-deoxyglucose, and glucosamine are known to be competitive inhibitors of approximately equal potency for glucose of yeast hexokinase (Ki approximately 0.7 mM for all three), metrizamide is a much weaker competitive inhibitor (Ki about 20 mM) of rat brain hexokinase than either 2-deoxyglucose or glucosamine (Ki about 0.3 mM for both). This indicates a greater active site specificity of rat brain hexokinase than of yeast hexokinase. Rat brain hexokinase activity is enhanced approximately threefold in the presence of 0.05, 0.2, and 0.8 mg/ml bovine serum albumin, while yeast hexokinase is only enhanced by 50% under these conditions. Despite the high Ki value for metrizamide, interference with glucose metabolism may occur whenever metrizamide is present in much higher concentrations than glucose. Myelography in humans is one such situation.

Animals↗