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PubMed1995

Molecular analysis of two hexokinase isoenzymes from Entamoeba histolytica.

The zymodemes, electrophoretic patterns of hexokinase, phosphoglucomutase and glucose phosphate isomerase isoenzymes, have been widely used to determine the pathogenicity of Entamoeba histolytica isolates. Although pathogenic and nonpathogenic forms of E. histolytica differ clearly in sequences of many homologous genes, a conversion between pathogenic and nonpathogenic zymodemes has been reported by several laboratories. To approach the question what might be the basis for the observed conversion, we examined the molecular biology of the hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzymes in pathogenic E. histolytica. We isolated two different cDNAs pHXK1 and pHXK2 coding for polypeptides with significant sequence similarity to hexokinases and deduced molecular masses of 49.8 kDa and 49.4 kDa. The two hexokinase sequences differed by 11% on the amino acid and by 8% on the nucleotide level. Expression of the cDNAs in Escherichia coli as nonfusion proteins gave two polypeptides with hexokinase activity. The recombinant Hxk1 and Hxk2 polypeptides comigrated with the more basic and more acidic isoforms of pathogenic amoebae in starch gel electrophoresis, as well as in low and high resolution isoelectric focussing gels. This identified the observed hexokinase isoenzymes of pathogenic E. histolytica as the products of two genes, hxk1 and hxk2.

Amino Acid Sequence↗
PubMed1984

Hexokinase in developing rabbit erythroid cells.

The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits separated by density centrifugation or by unit-gravity sedimentation. The specific activity of the enzyme was found to be about 150-fold higher in the basophilic erythroblasts as compared with the mature circulating erythrocytes. Most of the falls in hexokinase activity take place when the cell completes its final division and matures from the polychromatic stage to the orthochromatic stage. Concomitant with this strong decrease in enzyme activity, qualitative as well as quantitative changes in the hexokinase isozymic pattern become apparent. While in the basophilic and polychromatic erythroblasts the only hexokinase isozyme present is hexokinase type I, the orthochromatic cells also contain hexokinase Ib. This last isozymic form, which increases further at the reticulocyte stage, is also present in the circulating reticulocytes but not in mature red blood cells.

Anemia↗
PubMed1982

The comparative isozymology of vertebrate hexokinases.

1. Multiple hexokinase isozymes have been found in most vertebrates. Since each isozyme displays distinctive structural, kinetic and regulatory characteristics, the system qualifies as a useful probe for studies on molecular evolution. 2. At least seven types of chromatographic patterns of liver hexokinases have been observed in mammals. In contrast, each Class of lower vertebrates present only two or three distinct profiles. 3. Aves and higher Reptiles do not have the same hexokinase isozymes as other vertebrates. The nature of the differences is poorly understood. 4. Ontogenetic changes of liver hexokinase profiles are quite different in rat, chick and frog. 5. Structural comparisons of three vertebrate hexokinases having a molecular weight of approximately 100,000 suggest that those isozymes originated from a pre-vertebrate ancestor through gene duplication followed by fusion and further duplication events. Another hexokinase (the so-called glucokinase), with half the molecular weight, may have arisen either as the result of subsequent even splitting of the fused gene or, less probably, by divergence from a duplicated gene before the fusion event.

Amphibians↗
PubMed1985

Differences in the responses of brain cytosolic and mitochondrial hexokinases to three essential divalent metal ions.

Rat brain cytosolic and mitochondrial hexokinase activities were undetectable without added divalent cations. Mg2+ activated cytosolic (K0.5 of Mg2+ = 343 +/- 13 microM) and mitochondrial (K0.5 of Mg2+ = 183 +/- 8 microM) hexokinase in a concentration-related manner. The corresponding values for Mn2+ were 702 +/- 99 and 413 +/- 21 microM respectively. Ca2+, however, activated both forms of hexokinase poorly. In the presence of Mg2+, both Mn2+ and Cu2+ were more potent inhibitors of cytosolic hexokinase than mitochondrial hexokinase, whereas the inhibition of Cd2+ and Ca2+ did not show such selectivity. These results demonstrate that brain mitochondrial and cytosolic hexokinases differ significantly in their responses to divalent cations.

Animals↗
PubMed1988

Role of hexokinase in the regulation of erythrocyte hexose monophosphate pathway under oxidative stress.

Human erythrocytes overloaded with homogeneous human hexokinase (up to 15-times the activity of normal RBC) show almost unmodified rates of glucose metabolized in the HMP, however hexokinase-loaded RBC are able to metabolize 1.5 fold more glucose than controls through the HMP when an oxidizing agent like methylene blue (5 to 100 microM) is present. Similarly, RBC loaded with inactivating anti-hexokinase IgG (12 +/- 3% residual hexokinase activity) show HMP rates unchanged under resting conditions, but only 12% of the HMP rate found in normal controls under oxidative stress. These data provide clear evidence that the HMP rate under conditions of oxidative stress is controlled by hexokinase activity and suggest that RBC from patients with hexokinase deficiency are not able to increase the HMP rate under oxidative stress like erythrocytes from individuals with G6PD deficiency.

Erythrocytes↗
PubMedDate not supplied

Hexokinase translocation during neutrophil activation, chemotaxis, and phagocytosis: disruption by cytochalasin D, dexamethasone, and indomethacin.

Neutrophils expend large amounts of energy to perform demanding cell functions. To better understand energy production and flow during cell activation, immunofluorescence microscopy was employed to determine the location of the key metabolic enzyme hexokinase during various conditions. Hexokinase is translocated from the neutrophil's cytosol to its periphery in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and other activating stimuli, but not during exposure to the formyl peptide receptor antagonist N-tert-BOC-phe-leu-phe-leu-phe (Boc-PLPLP). Translocation was observed from 10(-6) to 10(-9)M fMLP. However, fMLP did not affect the intracellular distribution of lactate dehydrogenase. Hexokinase accumulated at the lamellipodium of cells exposured to an fMLP gradient whereas it localized to the phagosome after latex bead uptake. Thus, hexokinase is differentially translocated within cells depending upon the prevailing physiological conditions. Further studies noted that cytochalasin D, dexamethasone, and indomethacin blocked hexokinase translocation. Parallel regulation of reactive oxygen metabolite (ROM) production was shown. We speculate that hexokinase translocation participates in neutrophil activation.

Aminoquinolines↗
PubMed1998

Multiple crystal forms of hexokinase I: new insights regarding conformational dynamics, subunit interactions, and membrane association.

Hexokinase I is comprised of homologous N- and C-terminal domains, and binds to the outer membrane of mitochondria. Reported here is the structure of a new crystal form of recombinant human hexokinase I, which complements existing crystal structures. Evidently, in some packing environments and even in the presence of glucose and glucose 6-phosphate the N-terminal domain (but not the C-terminal domain) can undergo oscillations between closed and partially opened conformations. Subunit interfaces, present in all known crystal forms of hexokinase I, promote the formation of linear chains of hexokinase I dimers. Presented is a model for membrane-associated hexokinase I, in which linear chains of hexokinase I dimers are stabilized by interactions with mitochondrial porin.

Cell Membrane↗
PubMedDate not supplied

On the developmental properties and tissue interactions of hexokinase.

The interactions of the isozymes of hexokinase with cellular structure have been studied in the major tissues of the mouse during development. Overall, these data provide a clear indication that interactions between hexokinase and cellular structure are appreciable in all major tissues and at all stages of development, and an analysis of the isozyme status of the enzyme in both soluble and bound compartments has been effected. Further evidence in support of the already well documented interaction of hexokinase I to subcellular material in adult brain and kidney tissues is provided and extended to show that such interactions are extensive in both these tissues throughout development. In addition, evidence is provided that considerable hexokinase II activity is present in mouse foetal tissues in both the soluble and bound fractions and this isozyme is also shown to be the predominant "bound" form of the enzyme in adult skeletal muscle. By contrast, hexokinase III and IV are shown to be largely located in the cytosolic fraction of liver. The metabolic implications of these enzyme-structural interactions during development are discussed, as is the possibility of a functional linkage between hexokinase, which is bound to the mitochondria, and other enzymic components of the glycolytic sequence.

Animals↗
PubMed2003

Molecular and biochemical characterization of hexokinase from Trypanosoma cruzi.

The Trypanosoma cruzi hexokinase gene has been cloned, sequenced, and expressed as an active enzyme in Escherichia coli. Sequence analysis revealed 67% identity with its counterpart in Trypanosoma brucei but low similarity with all other available hexokinase sequences including those of human. It contains an N-terminal peroxisome-targeting signal (PTS-2) and has a calculated basic isoelectric point (pI = 9.67), a feature often associated with glycosomal proteins. The polypeptide has a predicted mass of approximately 50 kDa similar to that of many non-vertebrate hexokinases and the vertebrate hexokinase isoenzyme IV. The natural enzyme was purified to homogeneity from T. cruzi epimastigotes and appeared to exist in several aggregation states, an apparent tetramer being the predominant form. Its kinetic properties were compared with those of the purified recombinant protein. Higher K(m) values for glucose and ATP were found for the (His)(6)-tag-containing recombinant hexokinase. However, removal of the tag produced an enzyme displaying similar values as the natural enzyme (K(m) for glucose = 43 and 60 microM for the natural and the recombinant protein, respectively). None of these enzymes presented activity with fructose. As reported previously for hexokinases from several trypanosomatids, no inhibition was exerted by glucose 6-phosphate (G6-P). In contrast, a mixed-type inhibition was observed with inorganic pyrophosphate (PPi, K(i) = 0.5mM).

Amino Acid Sequence↗
PubMed1997

Purification and properties of buffalo (Bubalus bubalis) erythrocyte hexokinase.

Buffalo erythrocytes contain one isozyme of hexokinase that apparently lacks microheterogeneity as shown by chromatographic properties. A single protein band was detected by means of Western blotting using an antibody raised in rabbits against homogeneous rat brain hexokinase I. The native protein has a molecular weight of 200,000 +/- 2880 by gel filtration. Partial purification of erythrocyte hexokinase by a combination of several procedures, including affinity chromatography, which was previously applied successfully to the purification of other mammalian type I hexokinases, produced a partially purified enzyme that showed several contaminants after SDS-polyacrylamide gel electrophoresis. The affinity of buffalo erythrocyte hexokinase for glucose (K(m) = 0.012 +/- 0.001 mM) is lower than most other mammal hexokinases type I. It phosphorylates other sugars, with considerably higher K(m) values. This isozyme is able to use MgATP but does not use MgGTP, MgCTP or MgUTP. We used inhibition patterns, obtained with products to elucidate enzyme sequential mechanisms. Our results are clearly in agreement with a random sequential mechanism and in disagreement with an ordered sequential mechanism with either glucose or ATP as the obligatory first substrates. The ADP inhibition was of mixed type with both ATP and glucose as substrates.

Animals↗
PubMed2002

Sequencing, modeling, and selective inhibition of Trypanosoma brucei hexokinase.

For Trypanosoma brucei, a parasite responsible for African sleeping sickness, carbohydrate metabolism is the only source of ATP, and glycolytic enzymes are localized within membrane-bound organelles called glycosomes. Hexokinase, the first enzyme of the glycolytic pathway, was chosen as a target for selective drug design. We have cloned and sequenced the hexokinase gene of T. brucei. In parallel, we have synthesized several inhibitors. Kinetic analysis revealed differences in the binding mode of these compounds toward yeast and T. brucei hexokinases, while the m-bromophenyl glucosamide was found to be selective for T. brucei. The modeled structure of T. brucei hexokinase-inhibitor complex (using the crystal structure of the Schistosoma mansoni hexokinase as a template) allows us to propose a mode of action of this inhibitor for the trypanosome hexokinase and to account for the observed selectivity.

Adenosine Diphosphate↗
PubMed1983

Effect of inorganic phosphate on the reverse reaction of bovine brain hexokinase.

Kinetic studies were used to investigate the mode of brain hexokinase (EC 2.7.1.1, ATP:D-hexose 6-phosphotransferase) regulation by glucose 6-phosphate (glucose-6-P), ADP, and inorganic phosphate (Pi). A model for regulation of brain hexokinase by glucose-6-P and Pi had been proposed from initial-rate studies and binding experiments [Ellison, W. R., Lueck, J. D., & Fromm, H. J. (1975) J. Biol. Chem. 250, 1864-1871]. The results of the present investigation demonstrate that Pi is an activator of the brain hexokinase reaction when the reaction is studied in the nonphysiological direction. Evidence is presented which indicates that the back-reaction substrates and Pi can bind the enzyme simultaneously, and the suggestion is made that Pi binds to an allosteric site on the enzyme. These findings are in marked contrast to results obtained in the absence of ADP which convincingly demonstrate that glucose-6-P and Pi are mutually exclusive binding ligands for brain hexokinase. The kinetic data can be reconciled with the model for hexokinase regulation within the context of the well-established kinetic mechanism for brain hexokinase.

Adenosine Diphosphate↗
PubMed1999

Binding of rat brain hexokinase to recombinant yeast mitochondria: identification of necessary molecular determinants.

The association in vitro of rat brain hexokinase to mitochondria from rat liver or yeast (wild type, porinless, or expressing recombinant human porin) was studied in an effort to identify minimal requirements for each component. A short hydrophobic N-terminal peptide of hexokinase, readily cleavable by proteases, is absolutely required for its binding to all mitochondria. Mammalian porins are significantly cleaved at two positions in putative cytoplasmic loops around residues 110 and 200, as determined by proteolytic-fragment identification using antibodies. Recombinant human porin in yeast mitochondria is more sensitive to proteolysis than wild-type porin in rat liver mitochondria. Recombinant yeast mitochondria, harboring several natural or engineered porins from various sources, bind hexokinase to variable extent with marked preference for the mammalian porin1 isoform. Genetic alteration of this isoform at the C-, but not the N-terminal, results in a significant reduction of hexokinase binding ability. Macromolecular crowding (dextran) promotes a stronger association of the enzyme to all recombinant mitochondria, as well as to proteolytically digested organelles. Consequently, brain hexokinase association with heterologous mitochondria (yeast) in these conditions occurs to an extent comparable to that with homologous (rat) mitochondria. The study, also pertinent to the topology and organization of porin in the membrane, represents a necessary first step in the functional investigation of the physiological role of mammalian hexokinase binding to mitochondria in reconstituted heterologous recombinant systems, as models to cellular metabolism.

Animals↗
PubMed1997

Hexokinase binding to mitochondria: a basis for proliferative energy metabolism.

Current thought is that proliferating cells undergo a shift from oxidative to glycolytic metabolism, where the energy requirements of the rapidly dividing cell are provided by ATP from glycolysis. Drawing on the hexokinase-mitochondrial acceptor theory of insulin action, this article presents evidence suggesting that the increased binding of hexokinase to porin on mitochondria of cancer cells not only accelerates glycolysis by providing hexokinase with better access to ATP, but also stimulates the TCA cycle by providing the mitochondrion with ADP that acts as an acceptor for phosphoryl groups. Furthermore, this acceleration of the TCA cycle stimulates protein synthesis via two mechanisms: first, by increasing ATP production, and second, by provision of certain amino acids required for protein synthesis, since the amino acids glutamate, alanine, and aspartate are either reduction products or partially oxidized products of the intermediates of glycolysis and the TCA cycle. The utilization of oxygen in the course of the TCA cycle turnover is relatively diminished even though TCA cycle intermediates are being consumed. With partial oxidation of TCA cycle intermediates into amino acids, there is necessarily a reduction in formation of CO2 from pyruvate, seen as a relative diminution in utilization of oxygen in relation to carbon utilization. This has been assumed to be an inhibition of oxygen uptake and therefore a diminution of TCA cycle activity. Therefore a switch from oxidative metabolism to glycolytic metabolism has been assumed (the Crabtree effect). By stimulating both ATP production and protein synthesis for the rapidly dividing cell, the binding of hexokinase to mitochondrial porin lies at the core of proliferative energy metabolism. This article further reviews literature on the binding of the isozymes of hexokinase to porin, and on the evolution of insulin, proposing that intracellular insulin-like proteins directly bind hexokinase to mitochondrial porin.

Adenosine Triphosphate↗
PubMed2006

Mitochondrial hexokinases, novel mediators of the antiapoptotic effects of growth factors and Akt.

Cell survival has been closely linked to both trophic growth factor signaling and cellular metabolism. Such couplings have obvious physiologic and pathophysiologic implications, but their underlying molecular bases remain incompletely defined. As a common mediator of both the metabolic and anti-apoptotic effects of growth factors, the serine/threonine kinase Akt - also known as protein kinase B or PKB - is capable of regulating and coordinating these inter-related processes. The glucose dependence of the antiapoptotic effects of growth factors and Akt plus a strong correlation between Akt-regulated mitochondrial hexokinase association and apoptotic susceptibility suggest a major role for hexokinases in these effects. Mitochondrial hexokinases catalyse the first obligatory step of glucose metabolism and directly couple extramitochondrial glycolysis to intramitochondrial oxidative phosphorylation, and are thus well suited to play this role. The ability of Akt to regulate energy metabolism appears to have evolutionarily preceded the capacity to control cell survival. This suggests that Akt-dependent metabolic regulatory functions may have given rise to glucose-dependent antiapoptotic effects that evolved as an adaptive sensing system involving hexokinases and serve to ensure mitochondrial homeostasis, thereby coupling metabolism to cell survival. We hypothesize that the enlistment of Akt and hexokinase in the control of mammalian cell apoptosis evolved as a response to the recruitment of mitochondria to the apoptotic cascade. The central importance of mitochondrial hexokinases in cell survival also suggests that they may represent viable therapeutic targets in cancer.

Apoptosis↗
PubMed1969

Glucose metabolism in the mucosa of the small intestine. The effect of glucose on hexokinase activity.

1. The effect of three dietary components on hexokinase activity in the mucosa of rat small intestine was studied in vivo. Glucose, amino acids or an emulsion of monoglyceride with long-chain fatty acids were given by stomach tube to previously starved rats, and hexokinase activity was determined in the particle-free supernatant of mucosal homogenates. The formation of lactate from glucose and glucose 6-phosphate respectively was also measured. 2. When the three dietary components were given in isocaloric amounts, only glucose brought about an increase in hexokinase activity. 3. Intravenous injection of a similar amount of glucose to that given orally did not alter hexokinase activity. 4. An increase in the hexokinase activity of the particle-free supernatant prepared from mucosal homogenates was also observed after sacs of the small intestine of starved rats had been incubated in vitro in a medium containing glucose. Hexokinase activity increased to the values observed in corresponding preparations from fed rats, and this increase was strictly glucose-dependent.

Amino Acids↗
PubMed1984

Altered metabolic states do not change the intracellular distribution of hexokinase in Zajdela hepatoma ascites cells.

The distribution of hexokinase between bound and soluble forms was studied by digitonin fractionation of Zajdela hepatoma ascites cells maintained under various metabolic conditions. Addition of glucose to Zajdela cells respiring on endogenous substrates induces an immediate inhibition of respiration by 50-60% ( Crabtree effect), and a production of acid due to glycolysis. Acid production decreases abruptly after 60s to 50% of the initial rate. The ATP/ADP ratio is not altered by the addition of glucose or by different rates of glycolysis. The uncoupling agent carbonyl cyanide m-chlorophenylhydrazone decreases the ATP/ADP ratio by 10-fold in cells respiring on endogenous substrate, but has little effect on cells oxidizing glucose. Rapid fractionation of the cells under these various metabolic conditions revealed no change in the distribution of hexokinase. Approx. 75% of hexokinase is bound in all cases, in contrast with lactate dehydrogenase, 95% of which was in the soluble form. Longer-term incubations (to 20 min) revealed only slight (10-15%) increases in soluble hexokinase in cells incubated with glucose. Various metabolic inhibitors had little additional affect on the subcellular distribution of hexokinase. Thus a rapid release of hexokinase from mitochondrial membrane is not a mechanism by which glycolysis is regulated in rapidly growing Zajdela hepatoma.

Adenosine Diphosphate↗
PubMed1984

All hexokinase isoenzymes coexist in rat hepatocytes.

The cellular distribution of hexokinase isoenzymes, N-acetylglucosamine Kinase and pyruvate kinases in rat liver was studied. Hepatocytes and non-parenchymal cells with high viability and almost no cross-contamination were obtained by perfusion in situ of the liver with collagenase, with the use of an enriched cell-culture medium in all steps of cell isolation. Separation of hexokinase isoenzymes was done by DEAE-cellulose chromatography, and enzyme activities were measured by a specific radioassay. Cytosol from isolated hepatocytes contained high-affinity hexokinases A, B and C, in addition to hexokinase D. The last-mentioned represented about 95% of total glucose-phosphorylating activity. Only hexokinase A was found associated t the particulate fraction. Isolated non-parenchymal cells contained only hexokinases A, B and C. N-Acetylglucosamine kinase was measured with a specific radioassay and was found as a single enzyme form in both hepatocytes and non-parenchymal cells, with higher activities in the former. Pyruvate kinase isoenzyme L was present only in the hepatocytes and isoenzyme K only in the non-parenchymal liver cells, confirming that they are good cellular markers.

Animals↗