Particulate Hexokinase Research Articles

4-Phenyl butyric acid increases particulate hexokinase activity and protects against ROS injury in L6 myotubes

Hexokinase (HK) is the first enzyme in the glycolytic pathway and is responsible for glucose phosphorylation and fixation into the cell. HK (HK-II) is expressed in skeletal muscle and can be found in the cytosol or bound mitochondria, where it can protect cells against insults such as oxidative stress. 4-Phenyl butyric acid (4-PBA) is a chemical chaperone that inhibits endoplasmic reticulum stress and contributes to the restoring of glucose homeostasis. Aims: Here, we decided to investigate whether HK activity and its interaction with mitochondria could be a target of 4-PBA action. Main methods: L6 myotubes were treated with 1mM 4-PBA for 24, 48 or 72h. We evaluated HK activity, glucose and oxygen consumption, gene and protein expression. Key findings: We found that L6 myotubes treated with 4-PBA presented more HK activity in the particulate fraction, increased glucose consumption and augmented Glut4, Hk2 and Vdac1 mRNA expression. Moreover, 4-PBA prevented the deleterious effect of antimycin-A on HK particulate activity. Significance: Together, these results suggest a new role of 4-PBA in glucose metabolism that includes HK as a potential target of beneficial effect of 4-PBA.

Hexokinases of Tobacco Leaves: Changes in the Cytosolic and Non-Cytosolic Isozyme Complexes Induced by Tobacco Mosaic Virus Infection

Changes in the cytosotic (soluble) and the non-cytosolic (particulate) isozyme composition of hexokinases and in their properties were studied by ion exchange chromatography on DEAE cellulose after the subcellular fractionation both in the healthy and the tobacco mosaic virus (TMV) infected tobacco leaves. Three main isozyme complexes were obtained: one particulate fraction (the particulate hexokinase phosphorylating both glucose and fructose, EC 2.7.1.1), and two soluble fractions (the soluble hexokinase phosphorylating both the glucose and the fructose, and the soluble fructokinase, which phosphorylates primarily fructose, EC 2.7.1.4). The total fructokinase activities were nearly twice higher than the total glucokinase activities (188.6 % of glucokinase activity in healthy plants and 181.3 % in infected plants). The total particulate glucokinase activity was increased to 120.6 % and the fructokinase to 118.9 % in TMV infected tissue when compared with healthy control. The similar pattern of activity was observed for soluble hexokinase isozymes - the sum of soluble glucokinase activity was increased to 175.4 % and of fructokinase activity to 131.2 % in TMV infected tissue. The isozymes isolated both from the healthy control and TMV-infected leaves had the similar elution profiles, displayed Michaelis-Menten kinetics, showed the identical profiles of pH optima and were Mg2+ dependent with the highest enzyme activity at equimolar Mg2+ and ATP concentration.

Inhibition of Glycolysis and Respiration of Sarcoma-180 Cells by Echitamine Chloride

Malignant tumors are known to exhibit high rates of glycolytic activity leading to high production of lactic acid. Hence, neoplastic cells have elevated activity of enzymes responsible for glycolysis. Echitamine chloride, an indole alkaloid extracted from the bark of Alstonia scholaris, has been reported to have a highly promising anticancer activity against fibrosarcoma in rats. In the present study, the effect of echitamine chloride on energy metabolism of S-180 cells is investigated to have a better understanding on the mode of action of echitamine chloride. The effect of echitamine chloride on the mitochondrial and cellular respiration of S-180 cells was studied. Also, the effects on glucose utilization, pyruvate utilization and lactate formation were studied on whole S-180 cells and S-180 cell-free homogenate. The levels of glycolytic enzymes such as hexokinase and lactate dehydrogenase were estimated in which particular emphasis has been laid on hexokinase which occurs both in cytosolic and particulate forms in neoplastic cells. Hence the differential effect of echitamine chloride on the levels of total, cytosolic and particulate hexokinase has been investigated. In conclusion, echitamine chloride affects both cellular and mitochondrial respiration, leading to reduction of the cellular energy pool and thereby resulting in the loss of viability of S-180 cells.

Hexokinases of tobacco leaves: influence of plant age on particulate and soluble isozyme composition

Changes in hexokinase particulate and soluble isozyme composition and activities in leaves of 65- and 115-d-old tobacco plants were determined by ion exchange chromatography on DEAE cellulose. During plant ageing, the activities of glucose and of fructose phosphorylating isozymes of particulate hexokinase decreased to 9.9 and 9.2 % of initial value, respectively. The activity of soluble hexokinase decreased to a lesser extent: that of glucose phosphorylating isozyme to 49.8 % and of fructose phosphorylating isozyme to 37.8 %. The activity of soluble fructokinase isozyme dropped to 34.8 %. Thus also the ratio of particulate and soluble isozymes was dependent on the age of leaf tissue.

Hexose-6-kinases in germinating honey locust cotyledons: Substrate specificity of d-fructo-6-kinase

Extracts of the cotyledons of germinated honey locust ( Gleditsia triacanthos) seeds, which contain galactomannan as a reserve polysaccharide in the endosperm, were fractionated by chromatography and the fractions examined for the presence of a specific manno-6-kinase which could phosphorylate the d-mannose released by hydrolysis of galactomannan. One particulate hexokinase (the major hexose-6-kinase fraction) and two soluble hexokinase fractions (the minor portion), as well as a soluble fructo-6-kinase fraction, were initially separated. From chromatography, electrophoresis and kinetic studies, no evidence for a specific manno-kinase was obtained. This and the level and kinetic behaviour of the particulate hexokinase implicated it as the enzyme catalysing the phosphorylation of released d-mannose. The fructo-kinase activity was further separated into three fractions. Kinetic studies on one of these with native and synthetic substrates indicated that the structural requirements for the monosaccharide substrate were a β- d-anomeric 2-OH in the furanose ring, a 4-OH trans to the d-5-CH 2OH and a -CH 2OH substituent on C 2 ( trans to the 5-CH 2OH) which could be modified. The orientation of the hydroxyl on C-3 had only a limited effect.

Hexokinase receptors: preferential enzyme binding in normal cells to nonmitochondrial sites and in transformed cells to mitochondrial sites.

Hexokinase plays an important role in normal glucose-utilizing tissues like brain and kidney, and an even more important role in highly malignant cancer cells where it is markedly overexpressed. In both cell types, normal and transformed, a significant portion of the total hexokinase activity is bound to particulate material that sediments upon differential centrifugation with the crude "mitochondrial" fraction. In the case of brain, particulate binding may constitute most of the total hexokinase activity of the cell, and in highly malignant tumor cells as much as 80 percent of the total. When a variety of techniques are rigorously applied to better define the particulate location of hexokinase within the crude "mitochondrial fraction," a striking difference is observed between the distribution of hexokinase in normal and transformed cells. Significantly, particulate hexokinase found in rat brain, kidney, or liver consistently distributes with nonmitochondrial membrane markers whereas the particulate hexokinase of highly glycolytic hepatoma cells distributes with outer mitochondrial membrane markers. These studies indicate that within normal tissues hexokinase binds preferentially to nonmitochondrial receptor sites but upon transformation of such cells to yield poorly differentiated, highly malignant tumors, the overexpressed enzyme binds preferentially to outer mitochondrial membrane receptors. These studies, taken together with the well-known observation that, once solubilized, the particulate hexokinase from a normal tissue can bind to isolated mitochondria, are consistent with the presence in normal tissues of at least two different types of particulate receptors for hexokinase with different subcellular locations. A model which explains this unique transformation-dependent shift in the intracellular location of hexokinase is proposed.

Glucose catabolism in brain. Intracellular localization of hexokinase.

A major energy source in brain is glucose, which is committed to metabolism by hexokinase (Type I isozyme), an enzyme usually considered to be bound to the outer mitochondrial membrane. In this study, the subcellular location of hexokinase in brain has been rigorously investigated. Mitochondrial fractions containing hexokinase (greater than 500 milliunits/mg protein) were prepared by two different procedures, and then subjected to density gradient centrifugation before and after loading with barium phosphate, a technique designed to increase the density of the mitochondria. The gradient distribution patterns of both unloaded and loaded preparations show that brain hexokinase does not distribute exclusively with mitochondrial marker enzymes. This is particularly evident in the loaded preparations where there is a clear distinction between the peak activities of hexokinase and mitochondrial markers. The same observation was made when the mitochondrial fraction of either untreated or barium phosphate-loaded mitochondria was subjected to titration with digitonin. In fact, at concentrations of digitonin, which almost completely solubilize marker enzymes for both the inner and outer mitochondrial membranes, a significant fraction of the total hexokinase remains particulate bound. Electron microscopy confirmed that particulate material is still present under these conditions. Significantly, hexokinase is released from particulate material only at high concentrations of digitonin which solubilize the associated microsomal marker NADPH-cytochrome c reductase. Glucose 6-phosphate, which is known to release hexokinase from the brain "mitochondrial fraction" also releases hexokinase from this unidentified particulate component. These results on brain, a normal glucose utilizing tissue, differ from those obtained previously on highly glycolytic tumor cells where identical subfractionation procedures revealed a strictly outer mitochondrial membrane location for particulate hexokinase (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912). It is concluded that in brain, hexokinase has a greater propensity to localize at nonmitochondrial receptor sites than to those known to be associated with the outer mitochondrial membrane.

Hexokinases of tobacco leaves: Subcellular localization and characterization

Subeellular localization of the enzymes which phosphorylate hexoses was studied in photosynthesizing tobacco leaves by means of differential centrifugation and centrifugation in sucrose gradient. More than 80 % of the total hexokinase activity of leaf tissues were found to be associated with the particulate fraction of mitochondria; however, the ratio of the particulate hexokinase fraction to the soluble fraction was influenced by the extraction medium applied. The particulate hexokinases showed a high affinity to glucose (Km = 26.8 μM) and a relatively low affinity to fructose (Km = 17.6 mM). They had a broad pH optimum, because 81 % of the phosphorylating activity obtained for glucose and 75 % of the activity obtained for fructose occurred in pH range from 7.9 to 9.1 (Tris-HCl buffer). The hexokinases were Mg2+ dependent with the highest activity occurring at equimolar Mg2+ and ATP concentrations. Their activity was enhanced by KC1, NaCl, and (NH4)2SO4 at 30 to 120 mM concentrations.

Regulation of mitochondrial hexokinase in cultured HT 29 human cancer cells. An ultrastructural and biochemical study

The involvement of the mitochondrial bound hexokinase in aerobic glycolysis was investigated in two subpopulations of the HT 29 human colon cancer cell line: a poorly differentiated one with high aerobic lactate production (referred as undifferentiated or standard cells), and an enterocyte-like differentiated one with lower lactate production (referred as differentiated or Glc − cells). After mild digitonin treatment, 85% of the total cellular hexokinase activity remained in the particulate fraction in both cell types. In both cases mitochondria appeared to be tightly coupled but the Glc − cells exhibited a significantly higher oxidation rate in the presence of glucose. Electron microscopy of freeze-fractured cells revealed the absence of contacts between the two limiting mitochondrial membranes in the highly glycolytic standard cells, whereas the contacts were present in the Glc − cells. Furthermore, we investigated the functional relationship between bound hexokinase (as hexokinase-porin complex) and the inner compartment of mitochondria isolated from standard and Glc − HT 29 cells. In contrast to the differentiated cells the hexokinase in undifferentiated standard cells was not functionally coupled to the oxidative phosphorylation. This suggests that the high rate of lactate formation in neoplastic cells is not caused by an increase of particulate hexokinase activity but rather by a disregulation of the hexokinase-porin complex caused by the absence of contact sites between the two mitochondrial membranes. In agreement with this interpretation, the hexokinase-porin complex could be completely removed by digitonin treatment in standard HT 29 cells, while this was not possible in mitochondria from Glc − cells.

Enzymes of glucose metabolism in cultured human gliomas: neoplasia is accompanied by altered hexokinase, phosphofructokinase, and glucose-6-phosphate dehydrogenase levels.

The enzymes of glycolysis and selected enzymes of the pentose phosphate pathways were measured by fluorometric methods in extracts prepared from cultures of normal cortical human astrocytes and from cultures derived from low-grade (II) or high-grade (IV) gliomas. The hexokinase and phosphofructokinase levels of the low-grade glioma-derived line were not significantly different from those of the normal astrocyte cultures. However, the activities of hexokinase and phosphofructokinase were consistently and significantly increased in the high-grade glioma-derived lines. The activity of glucose-6-phosphate dehydrogenase was significantly decreased in all glioma-derived lines and by more than 90% in the high-grade-derived lines. Other enzymes of the glycolytic pathway were not significantly different from those of normal astrocytes, or they showed a variation inconsistently related to the neoplastic state. Glucose flux is not apparently regulated to a significant degree of hexokinase in glioma-derived lines, since the measured Vmax values are in substantial excess over the measured flux rates. Reversible binding of hexokinase to the particulate fraction was observed in both the normal astrocytes cultures and the high-grade glioma-derived lines. A twofold displacement of particulate hexokinase by ATP, ADP, 1-O-methylglucose, sorbitol-6-phosphate, and dibutyryl cyclic AMP was observed in the high-grade glioma-derived lines. The degree of displacement by various agents and the basal ratio of free/bound was not significantly different between the transformers and the nontransformants. The hexokinase from both the gliomas and the normal astrocytes was noncompetitively inhibited by the glucose analogue 2-deoxy-d-glucose. Phosphofructokinase activity is close to the observed glucose flux rates in both the normal astrocyte and the glioma-derived cultures. The phosphofructokinase activity of normal astrocytes is activated twofold or more by ADP, AMP, fructose-2,6-diphosphate, and Pi. However, these same ligands activate phosphofructokinase by less than twofold in a typical high-grade glioma-derived line. ATP, dibutyryl cyclic AMP, and citrate inhibit glioma and normal astrocytic phosphofructokinase, but the magnitude of the inhibition is much less than in the glioma-derived lines.

Regulation of glucokinase activity in the domestic fowl

The factors which regulate soluble and particulate glucokinase and hexokinase activity in the liver of domestic chickens has been investigated. Pretreatment with oral administration (via tube feeding) of glucose plus injection of insulin resulted in a significant increase in the activity of soluble (p less than 0.01) and particulate (p less than 0.01) glucokinase activity whereas fasting for 48 hours reduced glucokinase and hexokinase activity (p less than 0.01) in the particulate fraction only. Treatment of fed chickens for 2 weeks with thyroxine (50 micrograms: i.m. daily) plus triiodothyronine (50 micrograms) resulted in a marginal decrease (NS) in soluble glucokinase activity but significantly increased soluble hexokinase (p less than 0.05) activity. Thyroidectomized animals showed a decline in both soluble glucokinase (p less than 0.01) and hexokinase (p less than 0.025) activity. There was no effect of thyroid hormone manipulation on particulate glucokinase activity although there was a significant reduction in particulate hexokinase activity (p less than 0.05) in thyroidectomized birds. These data establish a physiological role for the glucokinase enzyme activity in avian carbohydrate metabolism and suggest that in contrast with the mammal, the particulate fraction is the more physiologically important enzyme.

Changes in the subcellular distribution of brain and heart hexokinase isoenzymes during alloxan diabetes

Changes in the subcellular distribution of hexokinase activity from three brain regions and heart were studied during alloxan induced diabetes. There was an overall decrease in the particulate hexokinase with an increase in the soluble form, after different time intervals of the onset of diabetes. Administration of insulin to the diabetic rats showed a partial counteraction of the enzyme changes. A possible regulation of brain hexokinase by metabolite changes is proposed

Hexokinase in human chorionic villi

The level, intracellular distribution, and isozymic pattern of hexokinase (EC 2.7.1.1) were determined on human chorionic villi obtained by trophoblast biopsy in the first trimester of pregnancy. About 50% of total hexokinase activity was found to be particle-bound and 96% of this in the overt form (i.e. assayable without the addition of detergents). Both soluble and particulate hexokinase show the same affinities for glucose but differ in the affinity for MgATP 2− and in their sensitivity to glucose 1,6-diphosphate inhibition. By chromatographic and kinetic methods the soluble hexokinase was found to be represented by isozymes I, II and traces of hexokinase III while most of the bound enzyme was hexokinase type I. These results provide evidence for the expression of at least three hexokinase isozymes in early human development and can partially explain the high rate of glucose utilization of the placenta.

Effect of 6-Aminonicotinamide on the activity of hexokinase and lactate dehydrogenase isoenzymes in regions of the rat brain

Changes in the activity of hexokinase and lactate dehydrogenase isoenzymes in the three brain regions and heart were studied in the 6-Aminonicotinamide-treated rats. Drug administration decreased the particulate hexokinase and lactate dehydrogenase activity, but increased the soluble hexokinase.

Age-dependent changes in glucose 1,6-bisphosphate levels and in the activities of glucose 1,6-bisphosphatase, and particulate hexokinase and 6-phosphogluconate dehydrogenase in rat skin

The levels of glucose 1,6-bisphosphate (Glc-1,6-P 2), the powerful regulator of carbohydrate metabolism, changed in rat skin during growth: Glc-1,6-P 2 increased during the first week of age, and thereafter was dramatically reduced during maturation. The activity of glucose 1,6-bisphosphatase, the enzyme that degradates Glc-1,6-P 2, changed with age in an invert manner as compared to the changes in Glc-1,6-P 2. These findings suggest that the age dependent changes in this enzyme's activity may account for the changes in intracellular Glc-1,6-P 2 concentration. The age-related changes in Glc-1,6-P 2 were accompanied by concomitant changes in the activities of particulate (mitochondrial) hexokinase and 6-phosphogluconate dehydrogenase, the two enzymes known to be inhibited by Glc-1,6-P 2. The activities of both these enzymes in the soluble fraction were not changed with age. The particulate enzymes were more susceptible to inhibition by Glc-1,6-P 2 than the soluble activities, which may explain why only the particulate, but not the soluble activities, correlated with the age-dependent changes in tissue Glc-1,6-P 2. These results suggest that the changes in particulate hexokinase and 6-phosphogluconate dehydrogenase resulted from changes in intracellular concentration of Glc-1,6-P 2. The marked reduction in Glc-1,6-P 2 during maturation, accompanied by activation of mitochondrial hexokinase and 6-phosphogluconate dehydrogenase, may reflect an enhancement in skin metabolism during growth.

Intracellular localization and properties of particulate hexokinase in the Novikoff ascites tumor. Evidence for an outer mitochondrial membrane location.

A detailed investigation concerned with localizing hexokinase in the Novikoff ascites tumor is presented. At least 50% of the total hexokinase activity was shown by differential and density gradient centrifugation techniques to be associated with tumor mitochondria. None of this activity was latent. Fractionation of isolated tumor mitochondria with digitonin revealed an outer membrane location for this enzyme. Treatment of tumor mitochondria with glucose 6-phosphate released about 80 to 85% of the hexokinase activity without disrupting the intermembrane compartment. This suggests that at least this proportion of the activity is bound to the outer surface of the outer membrane. Successive treatments did not remove the remaining hexokinase activity. At 30 degrees C, an incubation time of about 10 min with glucose 6-phosphate was required to achieve maximal release. No solubilization occurred at 0-4 degrees C. The isozymes derived from Novikoff mitochondria were identified by anion exchange chromatography as types I and III. Glucokinase activity was not detectable. Evidence is also presented which indicates that the hexokinase obtained from Novikoff mitochondria binds to the outer membrane of rat liver mitochondria. In contrast, the low endogenous hexokinase activity present in isolated liver mitochondria was found not to fractionate with outer membrane markers, but rather with contaminating microsomal membrane markers. Results described here provide the first direct evidence for the submitochondrial location of hexokinase in a tumor. They reveal an outer membrane location and an involvement of two hexokinase isozymes. Because these findings are characteristic of the hepatoma and not observed in control liver preparations, it is suggested that they may be very relevant to the general property of rapidly growing tumors to catabolize large amounts of glucose.

Hexokinases of spinach leaves

Two soluble hexokinases and a particulate hexokinase have been separated and partially purified from spinach leaves. One of the soluble hexokinases showed a high affinity for glucose ( K m = 63 μM) which was far greater than that for fructose ( K m = 9.1 mM). However, with saturating fructose the activity was twice that with saturating glucose. The particulate hexokinase showed kinetic properties similar to those of this soluble hexokinase. The second soluble hexokinase was distinct in that it was much more active with fructose than with glucose at all concentrations tested, although the K m values for these hexoses (210 μM and 71 μM respectively) were similar. The activity of this hexokinase was stimulated by the monovalent cations K + and NH 4 +.

Myo-Inositol metabolism in rat testis in response to streptozotocin-induced diabetes

The effects of streptozotocin-induced hyperglycemia on de novo myo-inositol biosynthesis in rat testis was examined. Testicular glucose and glucose 6-phosphate levels increased significantly 10 and 12 h after stretozotocin injection, respectively. However, testis myo-inositol content did not increase appreciably until 24 h following injection of the drug. Seventy-two hours after streptozotocin administration, testis myo-inositol levels were 2.7-fold higher in diabetic rats than in controls injected with citrate buffer. No changes were observed in the Specific activities of myo-inositol-1-phosphate synthase (EC 5.5.1.4) and 1- l- myo-inositol-1-phosphatase (EC 3.1.3.25). However, hyperglycemic rats displayed testicular glucose and glucose 6-phosphate levels approximately 4- and 2-fold in excess of control values, respectively. Insulin treatment of diabetic rats resulted in the lowering of plasma glucose, and testis glucose 6-phosphate to normal or below normal levels within hours. Inositol levels remained significantly elevated compared with control animals, although slightly lower than that observed for untreated diabetic rats. Streptozotocin diabetic rats had a significantly decreased testis cytosolic NAD + NADH ratio compared with control animals 72 h after injection. The potential role of testis hexokinase distribution in the regulation of glucose 6-phosphate and myo-inositol biosynthesis in normal and diabetic rats was investigated. No significant differences in testis hexokinase distribution or in the kinetic characteristics of the soluble and particulate hexokinase activities were observed. Testicular sperm counts in streptozotocin diabetic rats were not significantly different from control values.

Effect of fatty acids on rat brain particulate hexokinase.

Abstract— The effect of free fatty acids on rat brain particulate hexokinase was studied in vitro. Hexokinase bound with brain mitochondrial fraction was found to be sensitive to the action of free fatty acids, resulting in the solubilization of at least part of bound enzyme activity into the supernatant. The decrease of total enzyme activity observed at the highest free fatty acid concentration was probably due to the inhibition of hexokinase. The physiological consequence of hexokinase solubilization by low concentrations of free fatty acids, similar to that observed in vivo, is discussed in relation to activity changes of soluble and particulate enzyme forms demonstrated previously under hypoxic conditions.

Particulate and free hexokinase in fetal rat liver.

The association of hexokinase with particulate fractions of liver has been studied during the development of the rat. In the fetal rat liver about 50% of the total hexokinase is bound to the mitochondria. This proportion decreases gradually after birth. No association of hexokinase with mitochondria could be demonstrated in the adult rat liver. It is suggested that the binding of hexokinase to the mitochondria is a consequence of a property which is lost during development of the mitochondrial membranes. At least part of the hexokinase is rather tightly bound to the mitochondria. Glucose-6-phosphate or ATP do not solubilize the enzyme. The proportion of hexokinase bound to the mitochondria is independent of the metabolic state of the hepatocyte as exemplified by studies with inhibitors of oxidative phosphorylation, using the digitonin technique for the separation of particulate and cytosol fractions of isolated fetal rat hepatocytes.