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[Catalytic properties and isoenzyme composition of hexokinase in thyroid gland normal and neoplastic tissue].

Catalytic properties and isoenzyme composition of hexokinase were studied in extracts of normal thyroid, thyroid benign adenoma and thyroid carcinoma tissues from 23 patients. It was shown that the "cancer" hexokinase was several times more active and had lower Km (glucose) as compared with the enzyme from normal thyroid and benign tumor. Gel electrophoretic study revealed five hexokinase isoenzymes in both normal thyroid and its benign tumor. The hexokinase isoenzyme pattern in thyroid carcinoma was characterized by the "deletion" or distinctly decreased ratio of the "slowest" component. Studies on catalytic properties and isoenzyme composition of hexokinase are important as an additional test for the differentiation between benign and malignant tumors.

Adenocarcinoma↗
PubMed1994

Overexpression of hexokinase I in isolated islets of Langerhans via recombinant adenovirus. Enhancement of glucose metabolism and insulin secretion at basal but not stimulatory glucose levels.

Glucose metabolism and glucose-stimulated insulin secretion are thought to be controlled at the level of glucose phosphorylation in pancreatic islet beta-cells. In the current study we have investigated the importance of glucose phosphorylation by using recombinant adenovirus as a gene delivery system for isolated rat islets. Treatment of islets with a virus containing the cDNA encoding the Escherichia coli beta-galactosidase gene (AdCMV-beta GAL) resulted in efficiencies of gene transfer of 70.3 +/- 2.5 and 61.2 +/- 2.2% in two independent experiments. Treatment of islets with a virus containing the cDNA encoding rat hexokinase I (AdCMV-HKI) resulted in a 10.7-fold increase in immunodetectable hexokinase protein and a similar increase in enzyme activity. A large percentage of the overexpressed hexokinase activity was associated with a cell fraction enriched in mitochondria. These changes in enzyme level were accompanied by a 2-fold increase in insulin release and [5-3H]glucose usage at basal glucose concentrations (3 mM). The rate of glucose usage at 20 mM glucose and the magnitude of the insulin secretory response to this stimulatory level of the sugar were unchanged relative to control islets. Overexpression of hexokinase I in isolated islets therefore creates a phenotype of elevated basal insulin release similar to that seen in islets from obese and insulin-resistant mammals. The discrepancy between the large increase in hexokinase activity and the small increase in glucose usage and insulin release may indicate, however, that other steps in glucose metabolism become rate-limiting after only modest increases in glucose-phosphorylating activity.

Adenoviridae↗
PubMed1994

[Hexokinase isoenzyme II has a segment responsible for specific interaction of the enzyme with mitochondrial membranes].

It was found that the ability of rat skeletal muscle hexokinase isozyme II binding to mitochondrial membranes is realized in full in the presence of Mg2+, glucose and putrescine as adsorption reagent. The hexokinase-membrane complexes obtained through the use of these reagents displayed similar stability as could be judged from their dissociation under identical conditions: either in the presence of KCl used in high concentrations or under the effect of a specific solubilization reagent--glucose-6-phosphate used in physiological concentrations. Within the composition of enzyme-membrane complexes hexokinase had the same kinetic properties which differed, however, from those of the nonbound to mitochondria enzyme. The data obtained are discussed in relation to the hexokinase isozyme II domain responsible for the specific binding of the enzyme to mitochondrial membranes and termed as the "adsorption domain". The availability of this domain is postulated in terms of the concept on the adsorption mechanism of hexokinase isozyme II activity control in skeletal muscles.

Animals↗
PubMed1994

Insulin synthesis, secretory competence, and glucose utilization are sensitized by transgenic yeast hexokinase.

Glucokinase regulates insulin secretion by controlling the rate of glucose phosphorylation. In this report we utilize islets transgenic for high affinity yeast hexokinase to examine the role of glucose phosphorylation on other beta cell functions. Normal pancreatic islets responded to culture in low glucose by lowering insulin synthetic rates, becoming depleted of insulin and insulin mRNA, losing competence to respond to glucose with increased insulin secretion, and lowering glucokinase levels by one-half. In transgenic islets, increased high affinity hexokinase activity provided significant protection against reductions in all parameters of insulin synthesis and helped preserve the competence of beta cells to secrete insulin. The transgenic hexokinase also increased the rate of glucose utilization. These results demonstrate that glucose phosphorylation and presumably glucokinase mediate these glucose regulated responses. Of the parameters measured, only the change in glucokinase activity did not show an effect of the yeast hexokinase transgene. We also found that yeast hexokinase transgene expression was regulated 10-fold by glucose. This is the first demonstration of glucose inducibility of the insulin promoter in transgenic mice.

Animals↗
PubMed1976

The allosteric regulation of hexokinase C from amphibian liver.

A type C hexokinase (ATP:D-hexose-6-phosphotransferase EC 2.7.1.1) was partially purified from the liver of the frog Calyptocephalella caudiverbera. The enzyme is inhibited by glucose levels in the range of normal blood sugar concentrations. The extent of the inhibition by glucose depends on the concentration of ATP, being most marked between 1 and 5 mM ATP. Fructose, although a substrate, was not inhibitory of its own phosphorylation. The inhibitory effect of high glucose levels exhibited a strong, reversible pH dependence being most marked at pH 6.5. At pH 7.5 the inhibition by high glucose levels was a function of the enzyme concentration, the effect being stronger at high enzyme concentrations, whereas no inhibition was observed when assaying very diluted preparations. At all enzyme concentrations studied, high levels of glucose caused no inhibition at pH 8.5, whereas at pH 6.5 strong inhibition was always observed. Short times of photooxidation of hexokinase C as well as incubation with low concentrations of p-chloromercuribenzoate resulted in the loss of the inhibition by excess of glucose. Glucose-6-phosphate was found to be a strong inhibitor of hexokinase C but only at high glucose levels. The inhibitory effect of glucose-6-P follows sigmoidal kinetics at low (about 0.02 mM) glucose concentrations, the Hill coefficient being 2.3. The kinetics of the inhibition became hyperbolic at high (greater than 0.2 mM) glucose levels. These results suggest that the inhibition of hexokinase C by excess glucose is due to the interaction of glucose with a second, aldose-specific, regulatory site on the enzyme. The modification of the inhibitory effect by ATP, glucose-6-P, enzyme concentration, and pH, all of them at physiological levels, indicates a major role for hexokinase C in the regulation of glucose utilization by the liver.

Adenosine Triphosphate↗
PubMed1995

Rabbit brain hexokinase: evidence for the presence of two distinct molecular forms.

In mammalian brain tissue, most hexokinase is bound to the mitochondria and only a small amount of the enzyme is present in soluble form. In this study we report that, in rabbit brain, hexokinase is present in two distinct molecular forms, which we designated HKH and HKL, both of which are separable using hydrophobic interaction or anion-exchange chromatography. These two molecular forms can be detected when hexokinase is prepared at pH 7.4, whereas at pH 10.0 only the more hydrophobic form, HKH, is present. The two subtypes of hexokinase do not show significant differences in Km values for glucose and ATP, in Ki values for glucose-6-phosphate or in their molecular weights. HKH is able to rebind mitochondrial membranes, while HKL has lost this ability, suggesting that the hydrophobic peptide at the N-terminal has been removed. The susceptibility of the N-terminal peptide to proteolysis is completely inhibited by using antiproteolytic compounds, such as leupeptin or E-64. The results reported in this paper suggest that a cysteine protease, probably belonging to a the class of cathepsins, may be involved in the processing of bindable hexokinase to the non-bindable form in rabbit brain, and that the activity of this protease is pH-dependent.

Animals↗
PubMed1977

[Changes in the properties of hexokinase from hyaloplasm on binding with mitochondria].

It has been found that adenylic nucleotides at physiological concentrations solubilize mitochondrial hexokinase from muscles. The solubilizing effect of ATP is reversible in the presence of Mg2+ ions. It is shown that Mg2+ and glucose induce the transition of isoenzymes I and II of hexokinase from the free state to the bound one. Kinetic parameters of the free and bound forms of hexokinase have been determined. The stability of hexokinase has been found to vary, depending on its localization in the cell. Possible action mechanisms of reagents, exerting solubilizing and binding effects on the enzyme and the physiological role of hexokinase ability to exist in free and bound states are discussed.

Adenosine Triphosphate↗
PubMed1976

[Isoforms of human and animal tumor hexokinase].

In human brain tumors of neuro-ectodermal and meningo-vascular series, using the method of enzymoelectrophoresis and specific tetrazole blue staining, three isoforms of hexokinase were revealed, these differ from each other by their activity and electrophoretic mobility in agar gel. Three isoforms of hexokinase were also found in benign and malignant uterine tumors in females, in 22 A mice hepatoma and homologous intact tissues. Morever, in muscles and muscle tumors (MOP, CRM-1) of rats and of these animals embryos two isoforms of hexokinase were found. The increased rate of hexose phosphorylation in malignant uterine tumors of female patients and in blastomas of mice liver and rat muscles is associated with the increased activity of I and II isoforms of hexokinase. An analogous phenomenon is observed in muscles of rat embryos. On the other hand, the decreased activity of phosphotransferases in blastomas of human brain depends on a decrease in the activity of separate isoforms of hexokinase.

Animals↗
PubMed1997

Sugar induced cell death in yeast is dependent on the rate of sugar phosphorylation as determined by Arabidopsis thaliana hexokinase.

Sugars like glucose and fructose induce death of yeast cells within a few hours, in the absence of additional nutrients to support growth, while cells incubated in water remain viable for weeks. This sugar-induced cell death (SICD) by glucose and fructose required glucose or fructose phosphorylation since yeast cells deficient in hexose phosphorylation did not die. However, when hexose phosphorylation is restored by complementation with Arabidopsis thaliana hexokinase, the cells died. The affinity of A. thaliana hexokinase is about 400 times higher for glucose than for fructose, therefore, A. thaliana hexokinase was further utilized to study the role of hexose phosphorylation in SICD. The rate of SICD of hexokinase-deficient yeast cells expressing A. thaliana hexokinase was significantly slower in fructose than in glucose, indicating that SICD is determined by the rate of hexose phosphorylation. The significance of hexose phosphorylation and its role in SICD is discussed.

Journal Article↗
PubMed1985

Determination and characterization of hexokinase in thyroid cancer and benign neoplasms.

Hexokinase (ADP: D-hexose-6-phosphotransferase, EC 2.7.1.1) was studied in human thyroid carcinomas (n = 11), follicular adenomas (n = 32), and normal thyroid tissue (n = 21). The specific activity was significantly increased in carcinoma (0.163 +/- 0.083 U/mg protein) compared with normal tissue (0.030 +/- 0.010 U/mg protein) (P less than 0.001). Specific activities of follicular adenomas are rather heterogeneous, but when subdivided into three groups according to histopathologic criteria, a significant difference was found between follicular adenomas group I and II and follicular adenomas group III. A lesser cellular differentiation of adenomas is indicated by the lower degree or even absence of colloid production and follicle formation. A higher proliferation rate may be assumed on the grounds of the irregularities in outline, the often defective pseudocapsule, and signs of compression of the surrounding tissue. The highest specific activity in adenomas was found in the group with the highest proliferative activity, i.e., group III, whereas the lowest specific activities were found in adenomas with the lowest grade of proliferation, i.e., group I; the former was comparable with values found in carcinomas and the latter was comparable with values found in normal thyroid tissue. An interesting difference was found when the compartmentation of hexokinase was compared in carcinomas of different degree of differentiation. In papillary carcinomas a significantly lower proportion of hexokinase (HK) is present in the cytosol in comparison to follicular and undifferentiated carcinomas. In carcinomas more HK II and less HK I was found in comparison with normal thyroid tissue. In contrast hexokinase isozyme composition and compartmentation in adenomas were not different from normal thyroid tissue.

Adenoma↗
PubMed1981

Hexokinase "New Hyde Park": a low activity erythrocyte isozyme in a Chinese kindred.

Chronic hemolytic anemia in a 7-year-old Chinese boy was found to be associated with decreased activity of erythrocyte hexokinase (approximately 20% of appropriate control values). Alterations in kinetics, stability, pH optimum, or electrophoretic patterns could not be demonstrated with proband hexokinase, but subtle differences between maternal and paternal hexokinase characteristics suggested that two separate mutant isozymes were involved and that the proband was heterozygous for each. Comparisons with previously reported cases of hexokinase deficiency demonstrate the broad genetic heterogeneity that characterizes this disorder.

Anemia, Hemolytic↗
PubMed1981

Erythrocyte metabolism: kinetic and electrophoretic analyses of pig red cell hexokinase.

The mature erythrocyte of the pig has been observed to possess the slowest metabolic rate of any mammalian cell type. Previous studies in this laboratory suggested that the hexokinase isolated from these cells was inhibited by glucose in concentrations in excess of 0.2 mM. In the present study, the enzyme was isolated by utilizing DEAE-Sephadex A-50, ammonium sulfate precipitation, DEAE-cellulose (DE-52), and Sephadex G-100 gel-filtration. Studies on the hexokinase isolated from the pig mature erythrocyte by the above procedures revealed two distinct isozymes of hexokinase that do not behave kinetically and electrophoretically as those previously found in other mammalian red blood cells. The isozyme isolated from the erythrocyte of the young adult pig (less than six months of age) migrated at a slower electrophoretic rate than the one isolated from the adult pig (more than six months of age). Coupled with the observed difference in electrophoretic mobilities were changes in the apparent Km values as well as Vmax as a function of substrate concentration. In spite of the changes observed in relation to glucose, the apparent Km for Mg-ATP-2 was not altered during development. Diphosphoglycerate (DPG) was observed to be a "linear-mixed" inhibitor of both isozymes with respect to Mg-ATP-2. An experimental designed to determined the type of inhibition by DPG on the type I isozyme isolated from the horse erythrocyte revealed competitive inhibition with the Mg-ATP-2 site. Free Mg activated both isozymes in low concentrations (less than 2.5 mM) but inhibited the enzymatic activity as the concentration was elevated. The data suggest that both the young adult and the adult pig erythrocyte possess two distinct type III isozymes of hexokinase.

Adenosine Triphosphate↗
PubMed2006

Hexose-specificity of hexokinase and ADP-dependence of pyruvate kinase play important roles in the control of monosaccharide utilization in freshly diluted boar spermatozoa.

Incubation of boar sperm from fresh ejaculates in a minimal medium with 10 mM glucose induced a fast and intense activation of glycolysis, as indicated by the observed increases in the intracellular levels of glucose 6-phosphate (G 6-P) and ATP and the rate of formation of extracellular L-lactate. The effect of glucose was much more intense than that induced by fructose, sorbitol, and mannose. The greater utilization of glucose was related to a much greater sensitivity to hexokinase when compared with the other monosaccharides. Thus, the presence of 0.5 mM glucose induced total hexokinase activity in supernatants from sperm extracts of 1.7 +/- 0.1 mIU/mg protein, while the same concentration of both fructose, mannose, and sorbitol induced total hexokinase activity from 0.3 +/- 0.1 mIU/mg protein to 0.60 +/- 1 mIU/mg protein. Kinetic analysis of the total pyruvate kinase activity indicated that this activity was greatly dependent on the presence of ADP and also showed a great affinity for PEP, with an estimated Km in supernatants of 0.15-0.20 mM. Immunological location of proteins closely related to glycolysis, like GLUT-3 hexose transporter and hexokinase-I, indicated that these proteins showed the trend to be distributed around or in the cellular membranes of both head and midpiece in a grouped manner. We conclude that glycolysis is regulated by both the specific availability of a concrete sugar and the internal equilibrium between ATP and ADP levels. Furthermore, localization of proteins involved in the control of monosaccharide uptake and phosphorylation suggests that glycolysis starts at concrete points in the boar-sperm surface.

Adenosine Diphosphate↗
PubMed1993

Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.

Kinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families. The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium. The three-dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family. The second is the ribokinase family, of unknown three-dimensional structure, and comprises pro- and eukaryotic ribokinases, bacterial fructokinases, the minor 6-phosphofructokinase 2 from Escherichia coli, 6-phosphotagatokinase, 1-phosphofructokinase, and, possibly, inosine-guanosine kinase. The third family, also of unknown three-dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases. Each of the three families of sugar kinases appears to have a distinct three-dimensional fold, since conserved sequence patterns are strikingly different for the three families. Yet each catalyzes chemically equivalent reactions on similar or identical substrates. The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution. In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family. These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times. The flexible combination of active sites and three-dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function.

Amino Acid Sequence↗
PubMed1994

Porin proteins in mitochondria from rat pancreatic islet cells and white adipocytes: identification and regulation of hexokinase binding by the sulfonylurea glimepiride.

The binding of hexo-/glucokinase and glycerol kinase to mitochondria via the channel forming protein, porin, in pancreatic islet beta-cells and adipocytes, was recently proposed to participate in nutritional signaling, glucose sensing, and the control of high-energy phosphate distribution and oxidative phosphorylation. In this study we demonstrate that polyclonal antisera against purified rat liver porin recognize unique proteins in rat pancreatic islets, adipocytes, and RINm5F cells, each with an apparent M(r) about 2000 smaller than that of liver porin. Immunoblotting of subcellular fractions, the purity of which has been controlled by the distribution of marker proteins, revealed the mitochondrial localization of the cross-reacting proteins. Their enrichment with a method used for the purification of porin proteins, the characteristic behavior during isoelectric focusing, and the specific binding of rat liver hexokinase and glycerol kinase to phospholipid vesicles containing the purified cross-reacting beta-cell or adipocyte proteins strongly suggest their identity with mitochondrial porin. The subtle differences in the apparent M(r) and charge heterogeneity raise the possibility of the existence of porin isoforms expressed in a tissue-specific manner. Anti-porin antisera coimmunoprecipitated hexo-/glucokinase from rat insulinoma cell (RINm5F) and adipocyte mitochondria as determined by subsequent immunoblotting of the immunoprecipitates with polyclonal antisera against yeast hexokinase and rat liver glucokinase, respectively. This indicates that some rat pancreatic glucokinase (54 kDa) and liver hexokinase (102 kDa), respectively, is bound to mitochondrial porin. The major portion of the bound fraction is released from mitochondria after treatment with glucose 6-phosphate. Incubation of RINm5F and fat cells with the insulin releasing sulfonylurea drug, glimepiride (20 nM and 5 microM, respectively) for 30 min reduces the amount of hexo-/glucokinase associated with mitochondria and porin to about 50-30%. The reduced kinase binding activity of porin is preserved after isolation of porin from glimepiride-treated cells, reconstitution into phospholipid vesicles and assaying for glucose 6-phosphate inhibitable binding of rat liver hexokinase. The sulfonylurea tolbutamide (20 microM and 5 mM) is significantly less effective. The sulfonylurea-induced inhibition of hexo-/glucokinase binding to mitochondrial porin does not require glucose metabolism or Ca2+ influx into the cells. These data suggest that the sulfonylurea glimepiride, which is thought to inhibit the ATP-regulated K(+)-channel in beta-cells, may have, in addition, an intracellular site of action in pancreatic islet and adipocyte cells at the level of regulation of gluco-/hexokinase binding to mitochondrial porin.

Adipocytes↗
PubMed1994

Inactivation of rabbit red blood cell hexokinase activity promoted in vitro by an oxygen-radical-generating system.

Rabbit red blood cell hexokinase (EC 2.7.1.1) has been shown to be inactivated in vitro by incubating intact erythrocytes in the presence of an oxygen-radical-generating system represented by ascorbate and iron. It was interesting to note that among the glycolytic enzymes, only hexokinase was found to be susceptible to the action of oxygen radicals, suggesting that the loss of activity of this enzyme may be one of the first signals of cellular damage in rabbit red blood cells. This statement is supported by the fact that, under the experimental conditions used, we did not observe any significant plasma membrane lipid peroxidation nor intracellular proteolysis. Furthermore, mature erythrocytes are unable to synthesize hexokinase as well as other proteins de novo; therefore, the inactivation of this enzyme, which is both the first and one of the rate-limiting enzymes of the glycolytic pathway, could play an important role in determining metabolic impairment of red blood cells, with possible physiological implications. We also investigated the effect of various radical scavengers and antioxidants (glucose, vitamin E, dithiothreitol, flavonoids) which are able to influence the inactivation of hexokinase activity to different extents. Finally, under the experimental conditions used (90 min of incubation at 37 degrees C), we did not observe any difference in the hemolysis of rabbit red blood cells incubated in the presence or absence of ascorbate and iron (hemolysis was about 1% after 90 min of incubation), suggesting that the system used was able to furnish information about the cellular damage produced by oxygen radicals without provoking cell lysis.

Adenosine Triphosphatases↗
PubMed1997

Structure of the gene for type I hexokinase from rat.

Based on presumed analogy with the previously characterized gene encoding the Type II isozyme of rat hexokinase (Printz, R.L., Koch, S., Potter, L.R., O'Dougherty, R.M., Tiesinga, J.J., Moritz, S., and Granner, D. K., J. Biol. Chem. 268, 5209-5219, 1993), the locations of splice sites in the gene encoding the rat Type I isozyme of hexokinase have been determined by PCR amplification of intronic DNA. Sequences at the splice sites conform to the consensus sequence, with GT and AG being found at 5' and 3' ends of the introns, respectively. Sizes of exons 1 and 2 were determined directly while others were estimated based on identified splice sites and the previously determined cDNA sequence. These exon sizes were confirmed by PCR amplification, which gave products having sizes consistent with those of introns and exons predicted to be within the amplified sequence. Thus, it is unlikely that the gene encoding the Type I isozyme contains any introns not having analogs in the gene for Type II hexokinase. The deduced structure for the rat Type I hexokinase gene is therefore identical to that for the rat Type II isozyme, and spans over 51 kb. Six tandem repeat sequences of (AC/GT)n have been identified in the 5' flanking region and in introns 10, 11, 12, and 16; this is an unusually high frequency of tandem repeat sequences.

Animals↗
PubMed1998

Substrates of hexokinase, glucose-6-phosphate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase prevent the inhibitory response induced by ascorbic acid/iron and dehydroascorbic acid in rabbit erythrocytes.

Exposure of intact rabbit erythrocytes or erythrocyte lysates to ascorbic acid/FeCl3 in a glucose-free saline promoted a rapid decline in reduced glutathione and this response was paralleled by inactivation of hexokinase. Under the same conditions, the activity of the enzymes glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase did not show appreciablevariations in intact cells, but was severely inhibited in the cell-free system. Similar results were obtained by replacing ascorbic acid/FeCl3 with dehydroascorbic acid. In addition, both treatments effectively inhibited the activity of purified hexokinase as well as those of glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Further studies using the cell-free system indicated that the inhibition of enzyme activities elicited by either of the two treatments was effectively counteracted by the specific substrates of these enzymes. The fact that the hexokinase substrate glucose freely permeates the plasma membrane, unlike the substrates of glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphatedehydrogenase, explains the selective inhibition of hexokinase observed in intact cells. The above results also indicate that dehydroascorbic acid is an inhibitor of these enzymes and strongly suggest that it is at least in part responsible for the effects mediated by the cocktail ascorbic acid/FeCl3.

Animals↗