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PubMed1977

Studies on the functional significance of mitochondrial bound hexokinase in rabbit reticulocytes.

Mitochondria from rabbit reticulocytes contain about 50% of the total reticulocyte hexokinases. The proportion of mitochondrial hexokinases may be changed under different metabolic conditions. Mitochondrial bound and soluble hexokinases exhibit different kinetic properties (KMATP and glucose-6-phosphate inhibition). The respiratory rate of isolated reticulocyte mitochondria in the presence of glucose depends on the glucose-6-phosphate concentration, as the ADP generation by the endogenous hexokinases is strongly inhibited by glucose-6-phosphate. In the experimental system all intermediary states of mitochondrial respiration can be adjusted between the state of maximal activity (state 3 or active state) and the controlled or resting state (state 4) by different glucose-6-phosphate levels. The stationary levels of the extramitochondrial adenine nucleotides in this experimental system have been measured. The rate of mitochondrial respiration and ATP formation depends on the extramitochondrial ATP/ADP ratio. At ratios of about 10 and lower the mitochondria are in their maximum phosphorylation state, at higher ratios the mitochondrial ATP formation is controlled by the extramitochondrial ATP/ADP ratio. It is postulated that the close intercounnection between the mitochondrial hexokinase and the mitochondrial ATP forming system in reticulocytes is of funcitonal significance for mitochondrial-cytosolic interactions in rabbit reticulocytes and probably in other types of cells with mitochondrial hexokinases, too.

Adenosine Diphosphate↗
PubMed1978

[Tumorous origin of the hexokinase in human biological fluids].

Hexokinase of the endometrium and gastric mucosa is represented by 5 isoenzymes. The "simplification" of HK isoenzyme spectrum is characteristic of cancer tissue. So, in gastric cancer there is a disappearance of the "slowest" isoenzyme, while in malignant endometrium the "fastest" one was absent. Hexokinase isoenzymes of the serum were identical to those in the tumors in question, that indicates the tumor origin of the body fluid hexokinase. The latter was not observed in normal body fluids. The isoenzymic composition of hexokinase in uterine fibromyoma did not differ from that in normal tissues. If hexokinase appeared in the serum of these patients, its isoenzymic composition was similar to that in the normal uterus. The study on the hexokinase isoenzyme composition may be a valuable adjunct in establishing the differential diagnosis between benign and malignant tumors.

Body Fluids↗
PubMed1998

Light-induced photoactivation of hypericin affects the energy metabolism of human glioma cells by inhibiting hexokinase bound to mitochondria.

Glucose-dependent energy required for glioma metabolism depends on hexokinase, which is mainly bound to mitochondria. A decrease in intracellular pH leads to a release of hexokinase-binding, which in turn decreases glucose phosphorylation, ATP content, and cell proliferation. Thus, intracellular pH might be a target for therapy of gliomas, and a search for agents able to modulate intracellular pH was initiated. Hypericin, a natural photosensitizer, displays numerous biological activities when exposed to light. Its mechanism and site of action at the cellular level remain unclear, but it probably acts by a type II oxygen-dependent photosensitization mechanism producing singlet oxygen. Hypericin is also able to induce a photogenerated intracellular pH drop, which could constitute an alternative mechanism of hypericin action. In human glioma cells treated for 1 h with 2.5 microg/ml hypericin, light exposure induced a fall in intracellular pH. In these conditions, mitochondria-bound hexokinase was inhibited in a light- and dose-dependent manner, associated with a decreased ATP content, a decrease of mitochondrial transmembrane potential, and a depletion of intracellular glutathione. Hexokinase protein was effectively released from mitochondria, as measured by an ELISA using a specific anti-hexokinase antibody. In addition to decreased glutathione, a response to oxidative stress was confirmed by the concomitant increase in mRNA expression of gamma-glutamyl cysteine synthetase, which catalyzes the rate-limiting step in overall glutathione biosynthesis, and is subject to feedback regulation by glutathione. Hypericin also induced a dose- and light-dependent inhibition of [3H]thymidine uptake and induced apoptosis, as demonstrated by annexin V-FITC binding and cell morphology. This study confirmed the mitochondria as a primary target of photodynamic action. The multifaceted action of hypericin involves the alteration of mitochondria-bound hexokinase, initiating a cascade of events that converge to alter the energy metabolism of glioma cells and their survival. In view of the complex mechanism of action of hypericin, further exploration is warranted in a perspective of its clinical application as a potential phototoxic agent in the treatment of glioma tumors.

Anthracenes↗
PubMed1967

Effect of alloxan-diabetes on multiple forms of hexokinase in adipose tissue and lung.

Comparison has been made of the effect of alloxan-diabetes on the multiple forms of hexokinase (EC 2.7.1.1) in adipose tissue and lung. Types I and II hexokinase were distinguished in adipose tissue by their different stabilities to heat treatment, which made it possible to determine the activity of each form spectrophotometrically; additional confirmatory evidence was obtained from starch-gel electrophoresis. Type II hexokinase was markedly depressed in adipose tissue from alloxan-diabetic rats. Lung contained types I, II and III hexokinase, type I predominating. There was no significant change in the pattern of these multiple forms of hexokinase in lung from alloxan-diabetic rats. These results are discussed in relation to current ideas that the insulin-sensitivity of a tissue may be correlated with the content of type II hexokinase.

Journal Article↗
PubMed1984

Fructose is a good substrate for rat liver 'glucokinase' (hexokinase D).

Rat liver 'glucokinase' (hexokinase D) catalyses the phosphorylation of fructose with a maximal velocity about 2.5-fold higher than that for the phosphorylation of glucose. The saturation function is hyperbolic and the half-saturation concentration is about 300 mM. Fructose is a competitive inhibitor of the phosphorylation of glucose with a Ki of 107 mM. Fructose protects hexokinase D against inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid), and the apparent dissociation constants are about 300 mM in the presence of different concentrations of the inhibitor. The co-operativity of the enzyme in the phosphorylation of glucose can be abolished by addition of fructose to the reaction medium. Fructose appears to be no better as a substrate for the other mammalian hexokinases than it is for hexokinase D. It is proposed that the name 'glucokinase' ought to be reserved for enzymes that are truly specific for glucose, such as those of micro-organisms and invertebrates, and that liver glucokinase must be called hexokinase D (or hexokinase IV) within the classification EC 2.7.1.1.

Animals↗
PubMed1981

Hexokinase II of Pea Seeds.

A second hexokinase (EC 2.7.1.1) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed hexokinase II, had a high affinity (K(m), 48 micromolar) for glucose and a relatively low affinity (K(m), 10 millimolar) for fructose. The K(m) for MgATP was 86 micromolar. Mg(2+) was required for activity, but excess Mg(2+) was inhibitory. MgADP inhibited hexokinase II. The addition of salts of monovalent cations increased hexokinase II activity. Al(3+) was a strong inhibitor of the enzyme at pH 6.6 but not at the optimum pH (8.2). Citrate and 3-phosphoglycerate activated pea seed hexokinase II at pH 6.6, probably by coordinating with aluminum present as a contaminant in commercial ATP. The properties of hexokinase II are compared with those of the other three hexose kinases obtained from pea seed extracts. The possible role of these enzymes in plant carbohydrate metabolism is discussed.

Journal Article↗
PubMed1985

Cooperative interactions in hexokinase D ("glucokinase"). Kinetic and fluorescence studies.

Hexokinase D, also called hexokinase IV or glucokinase, is the isoenzyme characteristic of liver. In spite of its common name of glucokinase it phosphorylates also other sugars besides glucose; in particular, it phosphorylates fructose with similar specificity to that shown by the other hexokinases. Although hexokinase D is a monomeric protein it displays positive cooperativity with glucose and mannose. In contrast, the kinetic behaviour with 2-deoxyglucose and fructose is Michaelian. Mannose, fructose, 2-deoxyglucose and N-acetylglucosamine are competitive inhibitors of glucose phosphorylation and suppress the cooperativity. The cooperative behaviour can also be suppressed by the presence of glycerol at the assay medium at concentrations over 20%, with a decrease in the K0.5. Neither glycerol nor the inhibitors affect the monomeric state of the enzyme. Hexokinase D exhibits an intrinsic fluorescence at about 326 nm due to tryptophan residues. The binding of glucose to the enzyme enhances the native fluorescence by about 15%. A dissociation constant for glucose of about 3.5 mM was estimated; this value decreased to about 0.5 mM glucose in the presence of glycerol. These and other results are discussed on the basis of steady-state models which assume that hexokinase D exists mainly in one conformation state (EI) in the absence of ligands, and that the binding of glucose or mannose induces a conformational transition to a new conformation EII with higher affinity for the sugar substrates. It is postulated that differences in the velocities of the conformational transitions induced by the different sugar substrates give account of the differences in kinetic behaviour with the different sugar substrates.

Adenosine Triphosphate↗
PubMed1993

Inactivation of yeast hexokinase by Cibacron brilliant red 3B-A.

Procion or Cibacron blue dyes, containing polynuclear aromatic rings and mono- and dichlorotriazine nuclei, immobilized on dextran matrices, have been used for over a decade to purify diverse groups of enzymes by dye-ligand chromatography. Comparatively less attention has been paid to investigating the nature of molecular interactions between similarly constituted red dyes and various enzymes so as to ascertain their potential and thus justify their use in the purification of enzymes by dye-ligand chromatography. We investigated and found that Cibacron brilliant red 3B-A, a monochlorotriazine dye, inhibited phosphotransferase activity of yeast hexokinase. The dissociation constant, KD, and the rate of dye-enzyme complex formation, k3, were 120 microM and 0.1 min-1, respectively. The enzyme was protected from inactivation by sugar and nucleotide substrates. About 2 mol of the dye bound per mole of the enzyme. The chromophore of the dye showed absorption at 524 nm. The visible difference spectrum of increasing concentration of the dye and same concentrations of the dye plus a fixed concentration of hexokinase exhibited a maximum, a minimum, and an isosbestic point at 569, 501, and 512 nm, respectively. The difference spectrum of the dye and dye in 60% ethylene glycol showed a maximum and a minimum at 556 and 495 nm, respectively. The dye showed no visible difference spectrum in the presence of hexokinase modified at the active site by iodoacetamide, pyridoxal phosphate, and o-phthalaldehyde. Hexokinase modified by the dye coeluted with the unmodified enzyme during size-exclusion chromatography in the absence or presence of guanidinium hydrochloride. These results suggest that the dye interacts with the hydrophobic environment of the active site of the enzyme. Analysis of the kinetics of inhibition of hexokinase by model compounds and comparison of their computer-assisted three-dimensional representations with that of Cibacron brilliant red 3B-A suggest that 1-amino-8-naphthol-3,6-disulfonic acid may represent the minimum structure for the dye to bind.

Adenosine Triphosphate↗
PubMed1995

Residues putatively involved in binding of ATP and glucose 6-phosphate to a mammalian hexokinase: site-directed mutation at analogous positions in the N- and C-terminal halves of the type I isozyme.

Despite extensive sequence similarity between the N- and C-terminal halves of the Type I isozyme of mammalian hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1), they are functionally distinct, the C-terminal half being responsible for catalysis and the N-terminal half thought to play a regulatory role. We have examined the effects of several site-directed mutations on kinetic and regulatory properties of the rat Type I isozyme. Mutation of the C-terminal residues, Asp 532 to Asn, Arg 539 to Met, and Gly 896 or Gly 898 to Val, resulted in drastic loss of catalytic activity (< 10% of wild-type enzyme), consistent with previous suggestions that these residues are involved in binding of ATP. Mutation of the corresponding residues in the N-terminal half of the enzyme caused much less marked (> 50% of wild type), but significant, effects on activity which are presumed to result from subtle effects on conformation of the enzyme. Mutation of Lys 899 to Met resulted in an approximately 50% decrease in specific activity and an approximately fivefold increase in the Km for ATP, consistent with the view that Lys 899 participates in binding of ATP through electrostatic interactions with the phosphate sidechain. Cys residues corresponding to Cys 158 and Cys 606 of Type I hexokinase are found in other hexokinases that exhibit marked sensitivity to inhibition by the product, glucose 6-phosphate (Glc-6-P), but analogous residues are not found in hexokinases insensitive to Glc-6-P. However, this correlation appears to be coincidental since neither the mutation of Cys 158 or Cys 606 to Ala nor any of the other mutations examined abolished sensitivity of Type I hexokinase to inhibition by the Glc-6-P analog 1,5-anhydroglucitol-6-P or to antagonism of this inhibition by P(i).

Adenosine Triphosphate↗
PubMed1995

Application of a double isotopic labeling method to a study of the interaction of mitochondrially bound rat brain hexokinase with intramitochondrial compartments of ATP generated by oxidative phosphorylation.

gamma-Labeled ATP was produced by rat brain mitochondria utilizing [32P]Pi as substrate for oxidative phosphorylation. The 32P/14C ratio of Glc-6-P produced by the endogenous mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) using [U-14C]Glc as substrate was determined as a function of time after initiation of oxidative phosphorylation. This same ratio was determined for Glc-6-P formed by added yeast hexokinase using extramitochondrial ATP as substrate. The specific activity of ATP formed by oxidative phosphorylation was manipulated either by initiating the reaction with labeled Pi and subsequently adding excess unlabeled Pi or by initiating the reaction with unlabeled Pi and introducing the labeled substrate at a later time. The 32P/14C ratio of Glc-6-P formed by yeast hexokinase, reflecting the specific activity of ATP in the extramitochondrial space, was rapidly responsive to such manipulations, but the corresponding changes in the 32P/14C ratio of Glc-6-P produced by the endogenous hexokinase were markedly different. The results are consistent with the view that mitochondrially bound hexokinase does not utilize extramitochondrial ATP as substrate but rather is functionally coupled to a discrete intramitochondrial compartment of ATP produced by oxidative phosphorylation.

Adenosine Triphosphate↗
PubMed2002

Functional characteristics of hexokinase bound to the type a and type B sites of bovine brain mitochondria.

Hexokinase is released from Type A sites of brain mitochondria in the presence of glucose 6-phosphate (Glc-6-P); enzyme bound to Type B sites remains bound. Hexokinase of freshly isolated bovine brain mitochondria (Type A:Type B, approximately 40:60) selectively uses intramitochondrial ATP as substrate and is relatively insensitive to the competitive (vs ATP) inhibitor and Glc-6-P analog, 1,5-anhydroglucitol 6-phosphate (1,5-AnG-6-P). After removal of hexokinase bound at Type A sites, the remaining enzyme, bound at Type B sites, does not show selectivity for intramitochondrial ATP and has increased sensitivity to 1,5-AnG-6-P. Thus, the properties of the enzyme bound at Type B sites are modified by removal of hexokinase bound at Type A sites. It is suggested that mechanisms for regulation of mitochondrial hexokinase activity, and thereby cerebral glycolytic metabolism, may depend on the ratio of Type A:Type B sites, which varies in different species.

Adenosine Triphosphate↗
PubMed1997

Insulin-like growth factor I induces tumor hexokinase RNA expression in cancer cells.

Increased glycolysis is a characteristic of cancer cells. Though less efficient in energy production, it ensures continuous supply of energy and phosphometabolites for biosynthesis enabling metastatic and less vascularized cancer cells to proliferate even under hypoxic conditions. Since hexokinase is the first rate limiting enzyme in the glycolytic pathway, elevated levels of Type II like hexokinase in tumors are of great significance in this context. Under normal conditions insulin regulates expression of hexokinase Type II isoenzyme, which is predominantly expressed in muscle. On the other hand cancer cells overexpress insulin-like growth factors and their receptors which mimic many activities of insulin. This prompted us to examine a hypothesis that insulin-like growth factors may be responsible for overexpression of tumor hexokinase. Our experiments demonstrate that insulin-like growth factor I indeed induces hexokinase gene expression in a concentration and time dependent manner in two cancer cell lines we studied.

Blotting, Northern↗
PubMed1995

Compartmentation and metabolic parameters of mitochondrial hexokinase and creatine kinase depend on the rate of oxidative phosphorylation.

Oxidative phosphorylation in rat heart mitochondria was stimulated by the presence of hexokinase, by simultaneous operation of mitochondrial hexokinase and creatine kinase, or by mitochondrial hexokinase plus exogenously added phosphofructokinase. Under these conditions, 32Pi studies were conducted to estimate the extent of ATP compartmentation in the mitochondria in the vicinity of the active sites of hexokinase and creatine kinase. In all cases studied the extent of ATP compartmentation at 500 microM ATP concentration was no more than 12%. Within the same experimental design, the extent of ATP compartmentation increased with an increase in the rate of oxidative phosphorylation. The degree of ATP compartmentation depended on the relative location of the enzyme and inner mitochondrial membrane: it was maximal in the vicinity of the creatine kinase active sites and minimal for that of phosphofructokinase. The difference in the extent of ATP compartmentation in the neighborhood of the active sites of hexokinase and creatine kinase diminished with an increase in the rate of oxidative phosphorylation. We conclude that there is an ATP concentration gradient in the mitochondrial intermembrane space during oxidative phosphorylation, the minimum concentration being at the surface of the inner membrane. It was found that stimulation of oxidative phosphorylation led to a decrease in the apparent constants, Km (MgATP) and Vmax, for the two enzymes, however, to different degrees. Possible reasons for the change in kinetic parameters of the above enzymes are discussed.

Adenosine Triphosphate↗
PubMed1990

Similarities and differences between human and rat hexokinases type I.

The intracellular distribution and several properties of hexokinases type I purified to homogeneity from human placenta and rat brain were compared. The specific activity of the human enzyme was 190 +/- 5 U/mg protein; 140 +/- 5 U/mg protein that of the rat hexokinase. Comparative peptide mapping after limited tryptic digestion indicates a similar domain structure, however analogous experiments performed in the presence of substrates or effectors of the enzyme provide evidence of significant differences among hexokinases. Similarly, immunological studies with polyclonal and monoclonal antibodies while confirming some common epitopes also disclose important differences that cannot be expected on the basis of amino acid composition and of an in vivo identical function. These results are consistent with suggestions by several investigators that amino acid substitutions in mammalian hexokinases have occurred at a relatively fast rate during hexokinase type I evolution.

Amino Acid Sequence↗
PubMed1984

Comparison of rat brain and heart mitochondrial hexokinase solubilized by methohexital.

The purpose of the present investigation was to compare the solubilizing effect of methohexital on the mitochondrially bound hexokinase activity in brain and heart tissue of the rat. Experiments were performed using intact rats, the isolated perfused rat brain and heart as well as mitochondrial fractions from rat brain and heart tissue. It was shown that bound hexokinase activity was significantly solubilized by methohexital in brain tissue in vivo and in the isolated perfused rat brain but no effect was demonstrable in heart tissue. When mitochondrial fractions were incubated with methohexital in vitro, hexokinase activity was significantly solubilized from brain mitochondria but only slightly from heart mitochondria although glucose-6-phosphate was able to displace hexokinase also from heart mitochondria. The results suggest that mitochondrially bound hexokinase activity in brain tissue is particularly sensitive against the solubilizing effect of anesthetics. This effect could contribute to the sensitivity of brain function and metabolism against anesthetic drugs.

Animals↗
PubMed1982

Relationship between brain mitochondrial hexokinase and neuronal function: comparable effects of 2-deoxy-D-glucose and thiopental.

Mitochondrially bound brain hexokinase is solubilized by anesthetics and this effect has been suggested to contribute to anesthesia. In the present investigation the influence of the metabolic inhibitor 2-deoxy-D-glucose (2-DOG) was studied. An isolated rat brain preparation was used to avoid the contribution of peripheral reactions. Isolated rat brains were perfused for 45 min with media containing 4 mmol/l glucose, 10 mmol/l 2-DOG and/or 0.4 mmol/l thiopental. The EEG was monitored and acetylcholine, 2-DOG and its 6-phosphate, as well as the intracellular distribution of hexokinase activity were determined in brain tissue. Soluble hexokinase activity in brain cortex was enhanced by 2-DOG, as also by thiopental, and even more pronounced by both drugs used together, Results from in vitro experiments suggest that solubilization of mitochondrial hexokinase after 2-DOG is mediated by intracellularly accumulated 2-DOG-6-phosphate. 2-DOG produced a significant impairment of neuronal activity, revealing EEG patterns similar to those caused by thiopental anesthesia. Cortical acetylcholine levels were elevated by 2-DOG, as well as by thiopental, and again both drugs showed an additive effect when used in combination. This effect which may be the result of an inhibition of acetylcholine release, was also detectable in mice in vivo after 5 g 2-DOG/kg i.p., whereas the same dose of 3-O-methylglucose had no effect. The results provide further evidence that mitochondrial hexokinase may be involved in the relationship between cerebral metabolism and brain function.

Animals↗
PubMed1988

Hexokinase microheterogeneity in rabbit red blood cells and its behaviour during reticulocytes maturation.

Hexokinase in rabbit reticulocytes is present in two molecular forms (hexokinase Ia and Ib) separable by ion-exchange chromatography on DE-52 columns. By the use of ion-exchange HPLC we have been able to show that the isozymic form we previously called hexokinase Ia can be resolved into two peaks of activity one of which is (Ia) soluble, the other (Ia*) particulate. Hexokinase Ia* can be solubilized by detergents like saponine and Triton X-100 and disappears during 'in vivo' reticulocytes maturation. This new hexokinase microheterogeneity is not caused by different oxidized forms of the enzyme nor influenced by the presence of proteolytic inhibitors during lysate preparation.

Animals↗
PubMed2000

Sugar repression in the methylotrophic yeast Hansenula polymorpha studied by using hexokinase-negative, glucokinase-negative and double kinase-negative mutants.

Two glucose-phosphorylating enzymes, a hexokinase phosphorylating both glucose and fructose, and a glucose-specific glucokinase were electrophoretically separated in the methylotrophic yeast Hansenula polymorpha. Hexokinase-negative, glucokinase-negative and double kinase-negative mutants were isolated in H. polymorpha by using mutagenesis, selection and genetic crosses. Regulation of synthesis of the sugar-repressed alcohol oxidase, catalase and maltase was studied in different hexose kinase mutants. In the wild type and in mutants possessing either hexokinase or glucokinase, glucose repressed the synthesis of maltase, alcohol oxidase and catalase. Glucose repression of alcohol oxidase and catalase was abolished in mutants lacking both glucose-phosphorylating enzymes (i.e. in double kinase-negative mutants). Thus, glucose repression in H. polymorpha cells requires a glucose-phosphorylating enzyme, either hexokinase or glucokinase. The presence of fructose-phosphorylating hexokinase in the cell was specifically needed for fructose repression of alcohol oxidase, catalase and maltase. Hence, glucose or fructose has to be phosphorylated in order to cause repression of the synthesis of these enzymes in H. polymorpha suggesting that sugar repression in this yeast therefore relies on the catalytic activity of hexose kinases.

Deoxyglucose↗