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PubMed1999

Hexokinase I expression and activity in embryonic mouse heart during early and late organogenesis.

Hexokinase (HK) catalyzes the first step in glucose metabolism, that is, the conversion of glucose to glucose-6-phosphate (G6P). Four HK isoforms have been identified, of which HK-I is predominant in embryonic and fetal tissues. HK-I has been studied in preimplantation embryos and in fetal stages, but little is known about its activity or expression in the early postimplantation embryo. We evaluated HK-I expression, HK-I activity, and glycolytic metabolism in the embryonic mouse heart during early [gestational day (gd) 9.5] and late (gd 13.5) organogenesis. Immunohistochemistry demonstrated that HK-I is localized mainly in the heart at both stages, with stronger expression on gd 13.5. Densitometry after SDS-PAGE/western analysis confirmed higher immunodetectable HK-I protein levels in hearts on gd 13.5 vs gd 9.5. By contrast, RT-PCR demonstrated higher HK-I mRNA expression on gd 9.5 vs gd 13.5. Similarly, cardiac HK-I activity (conversion of glucose to G6P) and glycolysis (conversion of glucose to lactate) were higher on gd 9.5 than on gd 13.5. These results suggest a complex regulation of HK-I expression and activity in the embryonic heart during organogenesis, involving a change in the intrinsic activity of the enzyme with development. HK-I appears to play an important role in glucose metabolism during this critical stage of cardiogenesis.

Animals↗
PubMed2006

Evidence for intracellular spatial separation of hexokinases and fructokinases in tomato plants.

Four hexokinase (LeHXK1-4) and four fructokinase (LeFRK1-4) genes were identified in tomato plants. Previous GFP fusion studies indicate that the gene product of LeHXK3 is associated with the mitochondria while that of LeHXK4 is located within plastids. In this study we found that the enzyme encoded by the fructokinase gene LeFRK3 is also located within plastids. The presence of LeFrk3 enzyme in plastids raises the question of the origin of fructose in these organelles. The other three FRKs enzymes, LeFrk1&2&4, are located in the cytosol. Unlike LeFrk1&2&4, the two additional HXKs, LeHxk1&2, share a common membrane anchor domain and are associated with the mitochondria similar to LeHxk3. The difference in the locations of the cytoplasmic FRK and HXK isozymes suggests that glucose phosphorylation is confined to defined special intracellular localizations while fructose phosphorylation is less confined.

Amino Acid Sequence↗
PubMed2006

Function of interdomain alpha-helix in human brain hexokinase: covalent linkage and catalytic regulation between N- and C-terminal halves.

Human brain relies on a steady supply of glucose as the source of fuel, and type I hexokinase is the major isozyme governing the introduction of glucose to glycolysis in the brain. One unique regulatory property associated with type I isozyme is the alleviation of product inhibition by inorganic phosphate which binds to the N-terminal half, and the conformational change induced by inorganic phosphate must be propagated to the active site in the C-terminal half. With a single interdomain alpha-helix as the only covalent connection between the N- and C-terminal halves, the question arises as what role the interdomain alpha-helix plays at the interdomain signal transduction. Two mutants were constructed in an attempt to answer this question. The first mutant, A464P/E465G, with a helix breaker embedded in the interdomain alpha-helix had a smaller magnitude of phosphate alleviation than the wild type. The second mutant, with an insertion of seven additional residues between Gln 466 and His 467, had this phosphate relief property further diminished. Neither mutant showed dramatic changes nor the other kinetic properties. It is speculated that the interdomain alpha-helix is important for keeping the proper non-covalent contact so that transmission of the conformational changes across the N- and C-terminal half boundary can be achieved.

Amino Acid Substitution↗
PubMed1985

Binding and function of mitochondrial glycerol kinase in comparison with those of mitochondrial hexokinase.

Mitochondrial-bound glycerol kinase in rat brain was examined with reference to factors involved in the binding and significance of the binding in relation to ATP metabolism inside the mitochondria. The mitochondrial-bound glycerol kinase was solubilized with glycerol 3-phosphate or ADP, and the solubilized enzyme was rebound to mitochondria by addition of divalent cations. The rebinding was decreased by the presence of glycerol 3-phosphate, while was increased by glucose 6-phosphate. Positive correlation was found between the formation of glycerol 3-phosphate by mitochondrial-bound glycerol kinase and ATP content in mitochondria in experiments using various concentrations of succinate and ADP. On the other hand, glycerol 3-phosphate formation was inhibited by addition of inhibitors for mitochondria functions, such as oligomycin, dinitrophenol, cyanide, and atractyloside. Furthermore, formation of dihydroxyacetone phosphate from glycerol was proved, indicating the involvement of glycerol kinase in glycerol phosphate shuttle in combination with glycerol phosphate dehydrogenase. These findings are discussed in comparison with those of mitochondrial-bound hexokinase.

Adenosine Triphosphate↗
PubMed1985

Enhancement in the activities of mouse epidermal glucose-6-phosphate dehydrogenase, hexokinase, phosphofructokinase, and pyruvate kinase by 12-O-tetradecanoyl-phorbol-13-acetate.

The active ingredient in the tumor-promoting croton oil, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was shown to increase the activity of mouse skin epidermal glucose-6-phosphate dehydrogenase (+84%), hexokinase (+100%), phosphofructokinase (+158%), and pyruvate kinase (+101%). This increase in activity of these key enzymes of glucose metabolism occurred 2-8 h after TPA application and was due to a net increase in the enzyme content. This increase in the activity of the glycolytic enzymes, as well as the reported TPA-induced increase in the synthesis of RNA and DNA and cell proliferation, suggest that activation of the glycolytic pathway may be involved in the carcinogenic effects of tumor promoters.

Animals↗
PubMed1986

An examination of the in vivo distribution of brain hexokinase between the cytosol and the outer mitochondrial membrane.

These studies addressed the question of the in vivo distribution of rat brain hexokinase (HK), and whether physiologically relevant changes in the glycolytic rate are accompanied by changes in the distribution of HK. Homogenates of fresh tissue showed only 11-15% of the overt (assayable without added detergent) HK to be soluble (found in high-speed centrifugation supernatant fractions) when homogenization was begun within 15-20 s of sacrifice. Freeze-blown rat brain tissue also was used, coupled with a new technique wherein it was homogenized as it thawed in a buffered sucrose solution containing 1 mM EDTA. In tissue sampled 15 min (anesthetized) or 60 min (waking) after ip Nembutal injection (40 mg/kg), 23% of the overt HK and 79% of the total lactate dehydrogenase were soluble. The average phosphocreatine content of these and similar homogenates had decreased only 23% from in vivo levels, while ATP had decreased by 65%, due to the combined effects of a high level of endogenous ATPase, chelation of Mg2+ by EDTA, and the greater stability of Mg-ATP2- relative to Mg-ADP1-. These data indicated that the tissue experienced, at most, the equivalent of 6 s of complete ischemia prior to the completion of homogenization. Synaptosomes derived from rat and chicken cerebra were incubated at 37 degrees C in a physiological salt solution containing 10 mM glucose. Addition of veratridine has been shown to stimulate glycolysis and oxidative phosphorylation two- to threefold (H. T. Kyriazi and R. E. Basford (1986) J. Neurochem., in press), but did not alter the HK distribution, as 21% was found in the supernatant fractions of both control and veratridine-stimulated synaptosomes treated with digitonin. These results indicate that in brain tissue, large net movements of HK on and off the outer mitochondrial membrane do not occur, and thus play no role in the regulation of glycolysis.

Adenosine Triphosphatases↗
PubMed1988

A rare genetically determined electrophoretic variant of human leucocyte type III hexokinase.

Various lines of evidence from starch gel electrophoretic experiments demonstrate the existence of a genetically determined rare variant form of the type III isozyme of hexokinase (HK) in the leucocytes of a small percentage of the general human population. This enzymatically active variant (designated IIIS) migrates slightly, but significantly, slower than the common form (designated IIIF). In addition to finding various individuals with a two-banded pattern (heterozygotes containing both IIIS and IIIF), a finding reported previously by S. Povey, G. Corney, and H. Harris ((1975) Ann. Hum. Genet. 38, 407-415), we discovered one person homozygous for the variant phenotype. In close agreement with Povey et al., screening of 59 individuals at random indicated a gene frequency of about 0.017 for the IIIS allele, corresponding to a homozygous genotype for this allele that would be expected in about one of every 3500 individuals. Experiments involving the mixing of blood samples from the individual homozygous for IIIS with those homozygous for IIIF indicate that secondary in vitro changes, a possibility suggested by Povey et al., are not responsible for the appearance of the variant. This conclusion was supported by a demonstration of the specificity of the alteration in type III's mobility in comparison with the lack of alterations in any of the LDH isozymes, glucose-6-phosphate dehydrogenase, and various amido black-stainable proteins. These studies confirm the proposal for a genetically determined polymorphism of type III HK. No differences could be found between the total HK activity (according to spectrophotometric assays) of extracts from the subject homozygous for the variant and the activity from the homozygote for the common form, in terms of either their Km values for glucose or their heat stability properties. The similarity of Km values was supported by kinetic assays performed during staining of the individual forms on electrophoretic gels. Previous findings, reported elsewhere, of type III HK in RBC extracts were shown here to be attributable to contamination, by leucocytes, of the extracts. As a consequence of these studies, slight, but significant, amounts of type II-like HK were also discovered in leucocytes. Because our studies described above were completed in 1969, advantage was taken of the opportunity to test the HK pattern 17 years later from some of the same subjects. The patterns of the homozygotes for IIIS and for IIIF and the heterozygotes were found to be identical to the original ones, indicating no age-, environmental-, or other time-related changes that could explain the variation in type III HK.

Adult↗
PubMed1990

Differential scanning calorimetric study of rat brain hexokinase: domain structure and stability.

Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been studied by differential scanning calorimetry. In "high-ionic-strength" buffer (50 mM Tris-Cl, 0.5 mM EDTA, 10 mM monothioglycerol, pH 8.5), and assuming two-state behavior with calorimetric enthalpy equal to van't Hoff enthalpy, the endotherm could be deconvoluted into two transitions with Tm values of about 48 and 51 degrees C and enthalpies of about 109 and 112 kcal/mol, respectively. A similar endotherm was seen when glucose or glucose 6-phosphate was present, except that Tm values for both transitions were increased. The glucose analog, N-acetylglucosamine, had no observable effect on the endotherm, which is in agreement with previous studies indicating that this ligand, unlike glucose and glucose 6-phosphate, does not induce conformational changes that lead to increased stability of the enzyme. In "low-ionic-strength" buffer (5 mM Tris-Cl, 0.5 mM EDTA, 10 mM monothioglycerol, pH 8.5), the transitions were partially resolved even in the absence of ligands, with Tm values of about 49 and 55 degrees C. Due to difficulties with erratic baseline behavior under the low-ionic-strength conditions, enthalpies were not routinely determined, but these appeared to be similar to those seen in high-ionic-strength buffer. Also similar was the increase in stability, as reflected by the increase in Tm for both transitions, when glucose or glucose 6-phosphate was present. Correlation of these transitions with specific regions of the molecule was established by analysis of enzyme in which the domain corresponding to the first transition was selectively denatured by a partial scan in the calorimeter. Subsequent rescanning of these samples showed only the second transition, confirming the selective denaturation of the domain corresponding to the first transition and retention of the folded structure of the second domain. Discrimination between denatured (first transition) and undenatured (second transition) domains was based on the markedly increased susceptibility of the denatured region to tryptic digestion; regions of the molecule that retained their folded structure and resistance to proteolysis were identified by immunoblotting techniques using monoclonal antibodies recognizing epitopes having defined locations within the overall sequence. Based on this analysis, the first transition corresponds to unfolding of the C-terminal half of the molecule, with the second transition resulting from unfolding of the more stable N-terminal half. The order of unfolding could be reversed in the presence of ATP-Mg2+ and N-acetylglucosamine, conditions which have been shown to result in selective stabilization of the C-terminal domain.

Animals↗
PubMed1992

Epitopic regions recognized by monoclonal antibodies against rat brain hexokinase: association with catalytic and regulatory function.

Using direct and competitive epitope mapping methods, 23 monoclonal antibodies (Mabs) against rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) were divided into nine groups, each recognizing epitopes within defined surface regions of the N- or C-terminal domains; the latter have been associated with regulatory or catalytic functions, respectively. Reactivity of Mabs with the isolated domains was also studied. Based on the effect of various ligands on immunoreactivity, specific regions involved in ligand-induced conformational changes were identified. Adjacent epitopic regions, designated Regions F and G and located in the N- and C-terminal domains, respectively, were selectively affected by inhibitory hexose 6-phosphates (or analogs), marking these regions as being involved in transmission of the conformational signal from the regulatory N-terminal domain to the catalytic C-terminal domain. Consistent with this, the Ki for inhibition of the enzyme by the glucose 6-phosphate analog, 1,5-anhydroglucitol-6-phosphate, was markedly increased by Mabs binding in these regions, but unaffected by Mabs binding elsewhere in the molecule. Reactivity with Mabs recognizing conformationally sensitive epitopes in Region H of the C-terminal domain was greatly decreased by binding of substrate hexoses that induce closure of a cleft in the catalytic domain; selective recognition of the "open cleft" conformation, thereby preventing closure of the cleft required for progression of the catalytic cycle, can account for the marked decrease in Vmax that results from binding of these Mabs. Reactivity with Mabs binding to Region H was also decreased in the presence of inhibitory hexose 6-phosphates, implying that cleft closure was also induced by the latter; this is consistent with the suggestion that limitation of access to the C-terminal ATP binding site, resulting from cleft closure, is a factor in inhibition of the enzyme.

Amino Acid Sequence↗
PubMed1976

Purification and some properties of human erythrocyte hexokinase.

1. Human erythrocyte hexokinase (ADP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified 50 000--100 000-fold with a final specific activity of about 25--50 units/mg protein using gel-filtration, ion-exchange chromatography and affinity chromagraphy. 2. After isoelectrofocusing ofthe preparation one major protein band could be detected besides a minor band. THe isoelectric point of the major protein band was found to be 4.7. 3. After purification the enzyme could be stabilized in a medium containing inorganic phosphate, glucose, glycerol and mercaptoethanol. 4. The molecular weight was determined by gel-filtration and was found to be 132 000+/-8000. 5. The enzyme shows a broad pH optimum ranging from 7.0 to 8.4. 6. The kinetic behavior of the purified enzyme at 37 degrees C was somewhat different from the normal Michaelis-Menten kinetics due to its instability. The affinity constants were 0.048--0.080 mM for glucose and 0.57--1.0 mM for Mg-ATP. 7. The enzyme was specific for Mg- ATP as the nucleotide substrate. Mg-UTP, Mg-ITP,Mg-GTP and Mg-CTP were not converted to corresponding diphosphates. Several hexoses could be phosphorylated by the enzyme. Mannose could be phosphorylated at the same rate as glucose, although the affinity for the enzyme was lower (5m=0.60mM). Much lower rates and lower affinities were found with 2-deoxy-D-glucose (5m=1.0mM), D(+)-glucosamine (5m=4.5 mM) and fructose (5m=10 mM). N-acetyl-D-glucosamine , galactose andsorbose were not phosphorylated at all.

Drug Stability↗
PubMed1976

Kinetics of human erythrocyte hexokinase. Influence of temperature, ATP4- and magnesium ions.

Human erythrocyte hexokinase (EC 2.7.1.1) is inhibited competitively with respect to Mg-ATP2- by uncomplexed Mg2+ (Ki = 16--18 mM) and ATP4- (Ki = 1.6 mM). No real activation by low concentrations of Mg2+ could be detected and no allosteric behaviour was observed under the conditions tested. The temperature dependence of the enzyme was studied in relationship to the presence of Mg2+ or ATP4-. At equal concentrations of Mg2+ and ATP4- a break in the Arrhenius plot was observed at 27.5 degrees C, the higher temperature form of the enzyme having the lower activation energy. This break point in the Arrhenius plot was shifted to 36 degrees C in the presence of 5 mM Mg2+. A straight-line relationship was observed in the presence of 2.5 mM ATP4-. The Km for Mg-ATP2- showed a linear increase at temperatures over about 36 degrees C independent of the presence of Mg2+ or ATP4-. The nature of these phenomena is discussed.

Adenosine Triphosphate↗
PubMed1979

An essential arginyl residue in yeast hexokinase.

The inactivation of yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) by phenylglyoxal obeys pseudo first-order kinetics. Formation of a reversible enzyme-reagent complex prior to modification is suggested by the observed saturation kinetics. Loss of activity correlates with the incorporation of 1 mol of [14C]phenylglyoxal per mol 50 000 dalton subunit. No significant conformational change occurs concomitantly. Inactivation is attributable to modification of an arginyl residue. The pattern of protection by substrates and analogs favors an interaction of this essential residue with the terminal phosphoryl group of ATP or glucose 6-phosphate.

Adenosine Triphosphate↗
PubMed1980

Effects of free magnesium and alkali ions on the conformation and glucose-binding strength of yeast hexokinase isozymes.

Titrations of the tryptophan fluorescence of yeast hexokinase (ATP:D-hexose 6-phosphortransferase, EC 2.7.1.1.) isozymes P-I (A) and P-II (B) were performed with Mg2+, Li+, Na+ and K+ as titrant in absence and in presence of glucose, and vice versa, at pH 8.3 and 5.5 at 20 degrees C. Mg2+ quenches the fluorescence of surface tryptophan primarily and does so by producing a conformational change which alters the microenvironment of the tryptophan. For both isozymes Mg2+ exerts a specific ion effect, i.e. significantly larger than the ionic strength (I) effect, which enhances the glucose quenching by causing a conformation change which increases the glucose-binding constant. For the P-I isozyme glucose binding exhibits positive cooperativity at both pH 8.3 and 5.5 when the ionic strength (I) is low, i.e. significantly larger than the ionic strength (I) effect, which enhances the glucose quenching by causing a conformation change which increases the glucose-binding constant. For the P-I isozyme glucose binding exhibits positive sooperativity at both pH 8.3 and 5.5 when the ionic strength (I) is low, i.e. 0.04 or less, regardless of which of the above four cations is present. For P-II, however, glucose binding is non-cooperative at pH 8.3 regardless of I or the cation species and at pH 5.5 and low I with K+ or Mg2+ as the predominant cation present, but there is apparent negative cooperativity at pH 5.5 and low I when Na+ or Li+ predominates. These results are discussed in terms of known structural characteristics of the isozymes.

Binding Sites↗
PubMed1981

Separation and characterization of hexokinase I subtypes from human erythrocytes.

Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) type 1 from human erythrocytes exists in four electrophoretical distinct forms, termed Ia, Ib, Ic and Id in order of their increasing anodal electrophoretic mobility at pH 8.8. We were able to separate type Ia, Ib and Icd on phosphocellulose by using a discontinuous gradient elution. The three chromatographically distinct forms do not differ in their affinity constants for the substrates glucose and MgATP2-. In addition the inhibition by glucose 1,6-diphosphate does not differ significantly for all forms. However, the regulation of these inhibitions by inorganic phosphate is much less for type Ia compared to the other subtypes (P = 0.001). Aging of the red cells is accompanied by a relative increase of the proportion of type Ic and Ia, which is the less regulated form of the enzyme. This shift in electrophoretic and regulatory properties is argued to be due to a post-translational modification of the primary enzyme.

Adenosine Triphosphate↗
PubMed1988

Glucose metabolism via the Embden-Meyerhof pathway is not involved in ATP production during spore germination of bacillus megaterium QM B1551. A study with a mutant lacking hexokinase.

In order to investigate contributions by glucose metabolism via the Embden-Meyerhof pathway and that via the direct oxidation route to gluconate to initial ATP production during spore germination, respiratory activity and RNA synthesis were compared between the mutant lacking hexokinase and the parent spores of Bacillus megaterium QM B1551. We found that time courses of those metabolic events were almost identical between those spores, thus clearly indicating that NADH formed by a spore-specific enzyme glucose dehydrogenase (EC 1.1.1.47) is solely responsible for aerobic production of ATP at this stage.

Adenosine Triphosphate↗
PubMed1991

Protein kinase activity of rat brain hexokinase.

Hexokinase 1 (HK1) purified from rat brain exhibits protein kinase activity, including autophosphorylation and phosphorylation of histone H2A. This protein kinase activity is observed only in the absence of the HK1 carbohydrate substrate, glucose. Analysis of the ATP-binding domains of the mammalian HK1 protein sequences shows significant homology with other mammalian protein kinases.

Amino Acid Sequence↗
PubMed1984

Hexokinase, phosphofructokinase and pyruvate kinase isoenzymes in lymphocyte subpopulations.

In order to study the three regulator enzymes of glycolysis, hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK), in relation to lymphocyte maturation, lymphocytes of different origin were investigated. Lymphocytes from bone marrow, thymus, cord blood, adult peripheral blood and mitogen-stimulated lymphocytes were investigated. The enzyme activities were determined and the isozyme patterns were studied by means of electrophoresis, kinetic measurements and immunoprecipitation. The young lymphocytes from bone marrow and the mitogen-stimulated lymphocytes could be distinguished from the other lymphocytes by a higher residual HK activity in the presence of the inhibitor glucose-1,6-diphosphate. Peripheral blood T lymphocytes differed from non-T lymphocytes in the PK isozymes distribution. All the cells contained PK type K4 and the hybrid K3M. In T cells a smaller amount of the K isozyme was seen than in non-T cells. The PK residual activity in the presence of alanine was significantly higher in peripheral blood T cells than in non-T cells. Thymocytes are characterised by a larger amount of PFK M-subunits than peripheral blood T and non-T lymphocytes. The stimulation of PFK by the positive effector glucose-1,6-diphosphate was higher in thymocytes than in the peripheral blood lymphocytes.

Bone Marrow↗
PubMed1992

Evidence for multiple genes coding for the isozymes of hexokinase in the highly glycolytic AS-30D rat hepatoma.

We have compared Southern blots of rat hepatoma DNA probed with Types I, II and III hexokinase cDNAs isolated from normal rat tissues. Hybridization patterns show several fragments recognized by both the Type I and II clones while no resemblance is observed between the Type III probe and the other two isozymes. It therefore appears that the Type I-like and Type II-like hepatoma isozymes are coded for by similar yet separate genes, while a dissimilar third gene codes for the Type III-like isozyme. In addition, a loss of heterozygosity was detected at the Type III locus in the AS-30D hepatoma when compared to normal tissue. As only the Type II-like isozyme is highly expressed in highly glycolytic tumors, these data have implications for differential gene regulation between the tumor isozymes.

Alleles↗