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PubMed2001

Tubulin and microtubule are potential targets for brain hexokinase binding.

The metabolite-modulated association of a fraction of hexokinase to mitochondria in brain is well documented, however, the involvement of other non-mitochondrial components in the binding of the hexokinase is controversial. Now we present evidence that the hexokinase binds both tubulin and microtubules in brain in vitro systems. The interaction of tubulin with purified bovine brain hexokinase was characterized by displacement enzyme-linked immunosorbent assay using specific anti-brain hexokinase serum (IC(50)=4.0+/-1.4 microM). This value virtually was not affected by specific ligands such as ATP or glucose 6-phosphate. Microtubule-bound hexokinase obtained in reconstituted systems using microtubule and purified hexokinase or brain extract was visualized by transmission and immunoelectron microscopy on the surface of tubules. The association of purified bovine brain hexokinase with either tubulin or microtubules caused about 30% increase in the activity of the enzyme. This activation was also observed in brain, but not in muscle cell-free extract. The possible physiological relevance of the multiple heteroassociation of brain hexokinase is discussed.

Adenosine Triphosphate↗

PubMed1998

Hexokinase isoenzymes in normal and cirrhotic human liver: suppression of glucokinase in cirrhosis.

The activities of hexokinase isoenzymes I-IV (EC 2.7.1.1) and of N-acetylglucosamine kinase (EC 2.7.1.59) were determined in normal human liver and in alcoholic liver disease and primary biliary cirrhosis after FPLC fractionation of high-speed supernatants on Mono-Q with a linear NaCl gradient. In control human liver the hexokinase activities were: I, 3.6; II, 0.7; III, 3.5, IV, 4.8 (mUnits/mg supernatant protein). The activity of N-acetylglucosamine kinase was 8 mU/mg of protein. In alcoholic liver disease and primary biliary cirrhosis, the activity of hexokinase IV (glucokinase) was suppressed to less than 10% of control activity and the activity of hexokinase I was increased 3-fold. The activity of hexokinase II was increased approximately 7-fold in alcoholic liver disease. The activities of hexokinase III and N-acetylglucosamine kinase were unchanged in cirrhosis. Hexokinase III showed 50% substrate inhibition at 100 mM glucose as compared with 0.5mM glucose. The high activity of hexokinase III in human liver (approximately 50% of the low-Km activity and 70% of glucokinase activity) results in a significant underestimation of glucokinase activity as determined by the conventional spectrometric assay while the activity of N-acetylglucosamine kinase may contribute to an overestimation of glucokinase activity in the radiochemical assay. Furthermore glucokinase is dramatically suppressed in liver disease, which although partly compensated for by the increase in hexokinase I (and II), accounts in part for the well-known glucose intolerance of liver cirrhosis.

Chromatography↗

PubMed1998

In vitro and in vivo effects of heavy metals on mussel digestive gland hexokinase activity: the role of glutathione.

Hexokinase (E.C. 2.7.1.1), the enzyme responsible for glucose phosphorylation to G-6P, is inactivated by SH reagents and oxyradicals, and its inhibition has been involved in heavy metal toxicity in mammalian systems. In this work, the possibility that hexokinase activity could be affected by both heavy metal binding and oxidative stress conditions also in mussel tissues (Mytilus galloprovincialis Lam.) was investigated. The results obtained in vitro demonstrate that heavy metals inhibited digestive gland hexokinase (with Cd2+ > Cu2+ > Hg2+ > Zn2+ > Pb2+) and suggest a role for GSH in the protection against the heavy metal effects. Hexokinase activity was also reduced by addition of iron/ascorbate, indicating a susceptibility of the enzyme to metal-mediated oxyradical production. The effects of Cu2+ treatment (3 days, 40 micrograms l-1 per animal) on hexokinase activity and on the GSH/GSSG status were then evaluated in mussels exposed to a cycle of air exposure/reimmersion. In Cu-exposed mussels, a significant decrease in hexokinase activity and a parallel reduction in tissue GSH levels were observed, suggesting that the two effects of metal treatment could be related; however, hexokinase activity progressively recovered during air exposure and reimmersion, whereas the level of GSH showed a further decrease during air exposure followed by recovery after reimmersion. The in vitro results therefore indicate that mussel digestive gland hexokinase is susceptible to inactivation by heavy metal binding and suggest a role for GSH in the protection against the effects of heavy metals. The effects of copper were confirmed by the results obtained in vivo. The possible relationship between hexokinase activity and the level of GSH in the digestive gland of control and Cu-exposed mussels during air exposure and reimmersion are discussed, taking into account the balance between pro-oxidant and antioxidant processes at different stages of exposure.

Animals↗

PubMed1970

Glucose metabolism in the mucosa of the small intestine. Changes of hexokinase activity during perfusion of the proximal half of rat small intestine.

1. The effect of perfusion on the activities of hexokinase and lactate dehydrogenase was studied in the proximal half of the small intestine of fed and starved rats. 2. Perfusion of preparations from starved rats with a medium containing glucose caused a significant increase in hexokinase activity of the particle-free supernatant. The increase in activity was observed as early as 5min after the start of perfusion and persisted for up to 66min of perfusion. No increase in hexokinase activity of the particle-free supernatant was observed when a medium containing mannitol was used. As a further control, preparations from fed rats were perfused under the same conditions. With the medium containing glucose, the hexokinase activity of the particle-free supernatant remained unchanged during the first 15min of perfusion and thereafter fell gradually until, after 66min of perfusion, 73% of the original activity was retained. 3. The activity of lactate dehydrogenase in the particle-free supernatant prepared from the proximal half of the untreated small intestine of starved rats was significantly lower than in corresponding preparations from fed animals. However, it did not change significantly on perfusion with media containing either mannitol or glucose. 4. The distribution of hexokinase activity between total particulate fraction and particle-free supernatant was measured in preparations from starved rats after perfusion for 5-10min. In preparations that had not been perfused the ratio of hexokinase activity in total particulate fraction/particle-free supernatant was significantly higher in starved than in fed animals. After perfusion with a medium containing glucose, the total homogenate activity had not changed significantly, whereas the ratio of hexokinase activity in total particulate fraction/particle-free supernatant decreased significantly and approached the value obtained with fed animals. 5. The results agree with the view that the glucose-dependent increase of hexokinase activity in the soluble cell compartment as observed in vivo and in vitro in the intestinal mucosa of starved rats is brought about by a release of hexokinase activity from a particulate subcellular structure(s).

Animals↗

PubMed1978

Purification of the hexokinases by affinity chromatography on sepharose-N-aminoacylglucosamine derivates. Design of affinity matrices from free solution kinetics.

The purification is described of rat hepatic hexokinase type III and kidney hexokinase type I on a large scale by using a combination of conventional and affinity techniques similar to those previously used for the purification of rat hepatic glucokinase [Holroyde, Allen, Storer, Warsy, Chesher, Trayer, Cornish-Bowden & Walker (1976) Biochem. J. 153, 363-373] and muscle hexokinase type II [Holroyde & Trayer (1976) FEBS Lett. 62, 215-219]. The key to each purification was the use of a Sepharose-N-aminoacylglucosamine affinity matrix in which a high degree of specificity for a particular hexokinase isoenzyme could be introduced by either varying the length of the aminoacyl spacer and/or varying the ligand concentration coupled to the gel. This was predicted from a study of the free solution kinetic properties of the various N-aminoacylglucosamine derivatives used (N-aminopropionyl, N-aminobutyryl, N-aminohexanoyl and N-aminooctanoyl), synthesized as described by Holroyde, Chesher, Trayer & Walker [(1976) Biochem. J. 153, 351-361]. All derivatives were competitive inhibitors, with respect to glucose, of the hexokinase reaction, and there was a direct correlation between the Ki for a particular derivative and its ability to act as an affinity matrix when immobilized to CNBr-activated Sepharose 4B. Muscle hexokinase type II could be chromatographed on the Sepharose conjugates of all four N-aminoacylglucosamine derivatives, although the N-aminohexanoylglucosamine derivative proved best. This same derivative was readily able to bind hepatic glucokinase and hexokinase type III, but Sepharose-N-amino-octanoyl-glucosamine was better for these enzymes and was the only derivative capable of binding kidney hexokinase type I efficiently. Separate studies with yeast hexokinase showed that again only the Sepharose-N-amino-octanoylglucosamine was capable of acting as an efficient affinity matrix for this enzyme. Implications of these studies in our understanding of affinity-chromatography operation are discussed.

Animals↗

PubMed1988

Hexokinase type I multiplicity in human erythrocytes.

Hexokinase I in human erythrocytes exists in multiple molecular forms that differ in isoelectric points. By means of Western blotting and immunodetection of total glucose-phosphorylating activity by using an antibody raised in rabbit against homogeneous human placenta hexokinase I, a single protein band was detected. Identical results were also obtained by immunoaffinity chromatography of the partially purified enzyme. Separation of the three major hexokinase I subtypes (Ia, Ib and Ic) by h.p.l.c. ion-exchange chromatography and immunodetection following electrophoretic blotting confirmed that each hexokinase subtype showed the same apparent Mr of 112,000, which is the value obtained for the high-Mr hexokinase I from human placenta. Purification of erythrocyte hexokinase by a combination of several procedures including dye-ligand and affinity chromatography that were previously successfully applied to the purification of other mammalian hexokinases type I produced a 35,000-fold-purified enzyme that showed several contaminants after SDS/polyacrylamide-gel electrophoresis. Only one of these peptides was found to be recognized by anti-(hexokinase I) IgG, suggesting that proteolytic degradation does not occur and that hexokinases Ia, Ib and Ic have the same apparent Mr.

Chromatography, High Pressure Liquid↗

PubMed2004

Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria.

Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis. Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria. Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (Deltapsi(m)) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated Deltapsi(m), because glucose stabilized low Deltapsi(m) values in state 3. Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the Deltapsi(m) and in H(2)O(2) generation. The glucose analogue 2-deoxyglucose completely impaired H(2)O(2) formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H(2)O(2) generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented. Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellular glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.

Animals↗

PubMed1996

Unequal potency of transgenic yeast hexokinase on pancreatic beta cell metabolism and secretion.

Recent reports have proposed that sequence specific interactions between glucokinase and other beta cell proteins are important to glucokinase regulation of beta cell activity. We have previously reported enhancement of beta cell function by a transgenic hexokinase derived from yeast which has only 30% amino acid sequence homology to glucokinase. To test the functional significance of the amino acid sequence of islet glucokinase we have made a quantitative study of the effect of yeast hexokinase on beta cell glucose metabolism and insulin secretion. Transgenic and normal islets were assayed for hexokinase activity, glucose usage and insulin secretion. Most parameters were measured at six glucose concentrations between 0.5 and 20 mM glucose. Transgenic islet hexokinase activity measured in islet extracts exceeded normal islet hexokinase activity by 31 to 77 percent at different glucose concentrations. At all glucose concentrations tested the percentage increase in transgenic glucose metabolism greatly exceeded the percentage increase in transgenic hexokinase activity. The increase in transgenic glucose metabolism produced a proportional reduction in the threshold for glucose stimulated insulin secretion. However, yeast hexokinase had little if any effect on the first phase of insulin secretion. The finding that metabolism was very sensitive to yeast hexokinase but first phase secretion was not, supports recent proposals that hexokinase and glucokinase may be physically and functionally separated in the beta cell.

Amino Acid Sequence↗

PubMed1991

Modulation of hexokinase association with mitochondria analyzed with quantitative three-dimensional confocal microscopy.

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.

Animals↗

PubMed1996

Expression and activity of hexokinase in the early mouse embryo.

The maximal activity and Michaelis constant, KM, of hexokinase have been measured in the peri-implantation mouse embryo using an ultramicrofluorescence technique. In addition, transcript detection of the predominant isoenzyme hexokinase I has been determined in single preimplantation mouse embryos at successive stages of development using reverse transcriptase-mediated cDNA amplification. Maximal hexokinase activity decreased dramatically peri-implantation, from 0.97 +/- 0.19 nmol/microgram protein/h at the blastocyst stage to 0.31 +/- 0.05 nmol/microgram protein/h on day 6.5. The KM remained relatively low and constant over this period (0.23-0.39 mM), indicating the absence of the hexokinase type IV isoenzyme. The pattern of hexokinase activity resembled that of glucose consumption suggesting a possible regulatory role for the enzyme during this period of development. Hexokinase I mRNA was detected in the oocyte and all preimplantation stages of development. The blastocyst polymerase chain reaction (PCR) product, when cloned and sequenced was found to be 98% homologous with mouse tumour hexokinase I. Taken together, these data suggest that the hexokinase gene is not under transcriptional control during early mouse embryo development but plays a significant role in the regulation of glucose consumption. A role for hexokinase in the phosphate-induced inhibition of early embryo development is also proposed.

Animals↗

PubMed1990

Alloxan and ninhydrin inhibition of hexokinase from pancreatic islets and tumoural insulin-secreting cells.

Alloxan inhibited hexokinase activity in cytoplasmic fractions of transplantable radiation-induced rat islet cell tumours, ob/ob mouse pancreatic islets, rat liver and rat kidney. Half maximal inhibitory concentrations of alloxan were greater than those previously found for half maximal inhibition of pancreatic islet or liver glucokinase. D-glucose, preferentially the alpha-anomer, and D-mannose protected hexokinase activity against alloxan inhibition. 1,4-Dithiothreitol completely protected against and partially reversed the alloxan inhibition of hexokinase. The ability of various dithiols to reverse the inhibition of hexokinase by alloxan was dependent on the spacing between the SH (thiol) groups. Only dithiols with intermediate spacing between the SH groups were effective. Dithiols with two vicinal SH groups such as 1,2-dimercaptoethane and 2,3-dimercaptopropanol (BAL) and dithiols with more widely spaced SH groups such as 1,5-dimercaptopentane were ineffective. Thus a reaction of alloxan with two SH groups in the sugar binding site of the hexokinase with the formation of a disulfide bond may be involved in the reversible inhibition of the enzyme. Ninhydrin also inhibited hexokinase from all four tissues studied. The half maximal inhibitory concentrations of ninhydrin were lower than those of alloxan. Inhibition of hexokinase may be an important factor in the general cytotoxic action of ninhydrin. However, inhibition of pancreatic islet hexokinase is unlikely to be the initial event in the pancreatic B-cell toxic action of alloxan, even if inhibition of hexokinase by high concentrations of alloxan may contribute to the B-cell toxic action.(ABSTRACT TRUNCATED AT 250 WORDS)

Alloxan↗

PubMed1984

Saccharomyces cerevisiae mutants provide evidence of hexokinase PII as a bifunctional enzyme with catalytic and regulatory domains for triggering carbon catabolite repression.

A selection system has been devised for isolating hexokinase PII structural gene mutants that cause defects in carbon catabolite repression, but retain normal catalytic activity. We used diploid parental strains with homozygotic defects in the hexokinase PI structural gene and with only one functional hexokinase PII allele. Of 3,000 colonies tested, 35 mutants (hex1r) did not repress the synthesis of invertase, maltase, malate dehydrogenase, and respiratory enzymes. These mutants had additional hexokinase PII activity. In contrast to hex1 mutants (Entian et al., Mol. Gen. Genet. 156:99-105, 1977; F.K. Zimmermann and I. Scheel, Mol. Gen. Genet. 154:75-82, 1977), which were allelic to structural gene mutants of hexokinase PII and had no catalytic activity (K.-D. Entian, Mol. Gen. Gent. 178:633-637, 1980), the hex1r mutants sporulated hardly at all or formed aberrant cells. Those ascospores obtained were mostly inviable. As the few viable hex1r segregants were sterile, triploid cells were constructed to demonstrate allelism between hex1r mutants and hexokinase PII structural gene mutants. Metabolite concentrations, growth rate, and ethanol production were the same in hex1r mutants and their corresponding wild-type strains. Recombination of hexokinase and glucokinase alleles gave strains with different specific activities. The defect in carbon catabolite repression was strongly associated with the defect in hexokinase PII and was independent of the glucose phosphorylating capacity. Hence, a secondary effect caused by reduced hexose phosphorylation was not responsible for the repression defect in hex1 mutants. These results, and those with the hex1r mutants isolated, strongly supported our earlier hypothesis that hexokinase PII is a bifunctional enzyme with (i) catalytic activity and (ii) a regulatory component triggering carbon catabolite repression (Entian, Mol. Gen. Genet. 178:633-637, 1980; K.-D. Entian and D. Mecke, J. Biol. Chem. 257:870-874, 1982).

Alleles↗

PubMed1985

Regulation of hexokinase in cultured gliomas.

Positron emission computed tomographic (PECT) scanning studies have demonstrated that high grade gliomas exhibit increased 2-[18F]fluoro-2-deoxyglucose (18FDG) uptake compared to cerebral white matter and low grade gliomas. Hexokinase catalyzes the phosphorylation of glucose, as well as 18FDG and 2-deoxyglucose (2DG), thereby "trapping" these slowly metabolized analogues intracellularly. We hypothesize that a similar hexokinase-mediated uptake of glucose and glucose analogues occurs in vitro. Hexokinase activity was assayed in homogenates of tissue-cultured lines derived from high (IV) and low (II) grade gliomas and in fibroblasts derived from skin. With glucose as substrate, the maximal activity (Vmax) in the Grade IV lines was 200% of the activity found in the Grade II line, fibroblasts, and astrocytes; however, the Michaelis substrate affinity constant (Km) bore no relationship to tumor grade. With 2DG as substrate, the Vmax of all cell lines decreased, but the Grade IV lines still tended to have greater activity than the others. The Km values for 2DG were 5 times higher than those for glucose. Hexokinase is found in two subcellular compartments: an active form reversibly bound to mitochondria and a less active, cytosolic form. Up to 20% of the total hexokinase was found in the cytosol in all lines tested. High energy phosphate compounds (ATP, ADP, CTP, and others) displaced mitochondria-bound hexokinase, which increased the cytosolic form by 2-fold in the glioma lines, but fibroblast hexokinase distribution was unaffected. Our results suggest that: (a) high grade gliomas have increased hexokinase activity, which may explain the grade-related differences in 18FDG uptake observed by PECT scanning, and (b) human glioma hexokinases may be regulated by reversible subcellular compartmentation.

Cell Line↗

PubMed2005

Overexpression of hexokinase protects hypoxic and diabetic cardiomyocytes by increasing ATP generation.

Cardiac glucose metabolism is critical to hypoxic cardiac function and hypoxia is known to stimulate glucose metabolism. This increases generation of ATP when mitochondrial respiration is inhibited. In diabetes, cardiac glucose metabolism declines and this may contribute to diabetic cardiomyopathy. The first step in committing glucose to metabolism is glucose phosphorylation catalyzed by hexokinase. But the potential role of hexokinase in the hypoxic or diabetic heart is uncertain. This study is designed to assess the ability of hexo-kinase and elevated ATP to protect cardiomyocyte contractility from hypoxia and diabetes. We used cardiomyocytes from the transgenic mouse Mh, which has cardiac specific expression of yeast hexokinase, to investigate the importance of glucose phosphorylation in the myocyte response to hypoxia and diabetes. Cardiomyocytes were isolated from FVB control and Mh hearts to assess the effects of 2 h of hypoxia on myocyte contractility and ATP content. The protective effect of hexokinase on diabetes was assessed in myocytes from the OVE26 Type I diabetic mouse and in OVE26Mh diabetic mice that carry the hexokinase gene. Overexpression of hexokinase had no effect during aerobic culture, but during hypoxia, hexokinase improved ATP content by 44% and this restored contractility almost to normal levels. In myocytes from diabetic mice, tested under both aerobic and hypoxic conditions, the hexokinase gene significantly improved ATP content and this significantly improved contractility. These results demonstrate that elevating hexokinase activity can be beneficial to hypoxic or diabetic cardiomyocytes secondary to improving myocyte ATP levels.

Adenosine Triphosphate↗

PubMed1986

Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation.

In rabbit reticulocytes, the hexokinase (EC 2.7.1.1)-specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by a polyclonal antibody made in vitro shows that this maturation-dependent hexokinase decay is not due to accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system derived from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinae activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. Maximum ATP-dependent degradation was obtained at pH 7.5 in the presence of MgATP. MgGTP could replace MgATP with a relative stimulation of 0.90. 125I-Hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates that reach a steady-state concentration in 1 h, whereas the degradation of the enzyme is linear up to 8 h, suggesting that the formation of protein aggregates precedes enzyme catabolism. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mercaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugated to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP- and ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system of reticulocytes.

Adenosine Triphosphate↗

PubMed1984

Monoclonal antibodies against rat brain hexokinase. Effects on catalytic function and binding to the outer mitochondrial membrane.

A library of seven monoclonal antibodies has been prepared against rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1). Four of these antibodies, all of the IgG class, react with both native hexokinase and, to a more limited extent, with a ureadenatured S-carboxymethylated form of the enzyme. The other three antibodies, all of the IgM class, react readily with the denatured enzyme but are quite ineffective at binding native hexokinase. The monoclonal IgGs were further characterized with respect to their effects on certain functional properties of hexokinase. None had any detectable effect on catalytic properties, including inhibition by glucose 6-phosphate. One of the antibodies, designated 3C, totally blocked binding of the enzyme to the mitochondrial membrane, and significantly enhanced the release of the mitochondrially bound enzyme in either the absence or presence of glucose 6-phosphate, a ligand which promotes solubilization of mitochondrial hexokinase. It was concluded that the epitope recognized by 3C lies in, or immediately adjacent to, the region of the hexokinase molecule directly involved in interaction with the mitochondrial membrane. Two other IgGs, designated 1B and 2B, had only marginal effects on the binding of hexokinase to mitochondria, but were highly effective in preventing solubilization of the mitochondrially bound hexokinase by glucose 6-phosphate. Since these antibodies did not prevent binding of this ligand, as evidenced by the lack of an effect on inhibition, it is suggested that the effect of 1B and 2B on glucose 6-phosphate-induced solubilization is due to selective modification of the conformational changes that result from binding of glucose 6-phosphate. The monoclonal IgG designated 13 had no appreciable effect on either binding or glucose 6-phosphate-induced solubilization. The epitope for 13 is thought to lie in a "neutral" region of the hexokinase molecule, not involved in either catalytic or membrane-binding functions of the enzyme.

Animals↗

PubMed1984

Evidence for functional hexokinase compartmentation in rat skeletal muscle mitochondria.

Further studies of mitochondrially bound hexokinase have been carried out in order to elucidate the mechanism first proposed to increase efficiency of oxidative phosphorylation by the acceptor effect (Bessman, S. P. (1954) in Fat Metabolism (Najjar, V., ed) pp. 133-137, Johns Hopkins Press, Baltimore). During isolation of mitochondria, Mg2+ caused increased quantities of hexokinase to be bound or retained. This effect is concentration-dependent, saturable, and cannot be explained by Mg2+-linked activation or stabilization. Rebinding of hexokinase to isolated mitochondria also shows a similar dependence on Mg2+. When added to a homogenate made without it, Mg2+ could not bind the same amount of hexokinase to the mitochondria as could be observed when Mg2+ had been included in the homogenizing medium from the start. Using mitochondria prepared with Mg2+ in order to bind hexokinase to the largest extent possible, we have demonstrated that as in the case of mitochondrial creatine phosphokinase, a compartment exists that permits more efficient production of glucose 6-phosphate during mitochondrial respiration--the hexokinase acceptor effect. This effect probably results from a favorable positioning of the active site of hexokinase, perhaps within the intermembrane space, providing a diffusion-favorable situation. Thus, newly synthesized ATP transported through the inner membrane supplies substrate to hexokinase with greater efficiency than that of ATP which must pass through the outer membrane by diffusion from the medium. These observations lend support to proposals that in vivo modulation of the soluble particulate distribution of hexokinase by hormones or by metabolites may be physiologically necessary and important.

Adenine Nucleotides↗

PubMed1981

Energy metabolism of tumor cells. Requirement for a form of hexokinase with a propensity for mitochondrial binding.

Rat liver cytoplasm (postnuclear supernatant) has a low aerobic glycolytic rate in the presence of added glucose, ATP, ADP, Pi, and NAD+, whereas cytoplasm from Ehrlich ascites tumor cells exhibit a high aerobic glycolytic rate which is typical of rapidly proliferating tumor cells. Tumor mitochondria, unlike liver mitochondria, contain bound hexokinase which constitutes about 70% of the total cellular hexokinase activity. The high aerobic glycolytic rate of Ehrlich tumor cytoplasm is reduced markedly if the mitochondria are removed and can be restored almost completely upon addition of the hexokinase-containing tumor mitochondria to tumor cytosol (postmitochondrial supernatant). Addition of tumor mitochondria to liver cytosol can enhance its glycolytic rate to levels approaching those of tumor cytoplasm, whereas added liver mitochondria are without effect on the already low glycolytic rate of liver cytosol. Addition of tumor mitochondria to tumor cytosol increases its glycolytic rate to the level of tumor cytoplasm, as mentioned above, but liver mitochondria added to tumor cytosol actually depress its glycolytic rate to the level of liver cytosol. The stimulatory effect of tumor mitochondria on liver cytosol can be ascribed to its associated hexokinase activity since hexokinase specifically removed from mitochondria of tumor cells can also enhance the glycolytic rate of liver cytosol. The depressing effect of added liver mitochondria on tumor cytosol glycolysis suggests that liver mitochondria can compete more effectively than tumor mitochondria for a common intermediate and/or cofactor. Examination of 12 different tumor cell lines revealed that only those which reached maximum size in 1 month or less, and which have elevated glycolytic activities, had detectable mitochondrially associated hexokinase activity. The studies reported here describe resolution and reconstitution of tumor cytoplasm, supplementation of cytosol with intact mitochondria or mitochondrial hexokinase, and a survey of mitochondrial hexokinase content in various tumors, and provide strong evidence for the view (Bustamante, E., and Pedersen, P. L. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 3735-3739) that a form of hexokinase with a propensity for mitochondrial binding plays a key role in the high aerobic glycolysis of cancer cells.

Aerobiosis↗