Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.1.1 (hexokinase)
 5,274 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of ATP to immobilized firefly luciferase, the onset of light production is slower than that observed with the soluble enzyme, due to a slower diffusion of ATP to the immobilized enzyme. With immobilized pyruvate kinase, a relative accumulation of ATP inside the beads is demonstrated, as measured with coimmobilized firefly luciferase. The accumulation of product (ATP) is enhanced when the bead suspension is not stirred. This ATP in the beads is relatively inaccessible to soluble hexokinase added to the bulk medium. Similarly, a rapid ATP depletion in the microenvironment of immobilized hexokinase is demonstrated. This microscopic event is kinetically distinguishable from the slower macroscopic depletion of substrate in the bulk medium. The rate of depletion in the microenvironment depends on the local activity of the immobilized enzyme but not on the total amount of enzyme in suspension, as does the macroscopic phenomenon. The theoretical principles for the interaction of diffusion and catalysis in these systems are briefly summarized and discussed. These results are relevant to various molecular mechanisms proposed for membrane-bound enzyme action and regulation, derived from macroscopic kinetic measurements assuming a negligible diffusion control.
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PMID:Continuous monitoring of adenosine 5'-triphosphate in the microenvironment of immobilized enzymes by firefly luciferase. 365 23

It was found that in the presence of Mg2+ (pH 7.5) rat skeletal muscle hexokinase isozyme II is firmly adsorbed on mitochondrial and artificial phospholipid membranes (lecithin liposomes). In both cases the adsorption isotherm has similar quantitative and qualitative characteristics, which points to the absence of specific binding sites on the membranes. Under these conditions, immobilization of hexokinase on various membranes is concomitant with similar changes in the enzyme stability upon storage as well as with the pH-dependence of the enzyme activity. It was demonstrated that the bound hexokinase form has a greater value of V, an increased affinity for glucose and a decreased sensitivity to the inhibitory action of glucose-6-phosphate as compared to the free form. Besides, this form is in a greater degree subjected to the inhibitory influence of ADP with respect to glucose. In this case, the enzyme affinity for ATP and the Ki value for ADP with respect to ATP is practically the same both for the free and membrane-bound forms. The data obtained suggest that the phospholipid component of mitochondrial membranes participates in the enzyme binding in the presence of Mg2+. It was assumed that the model system used in the present study, i.e., hexokinase-Mg2+-liposomes, may be successfully used for the analysis of an adsorption mechanism of regulation of hexokinase activity in the cell.
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PMID:[Interaction of hexokinase II isoenzyme from rat skeletal muscles with lecithin liposomes]. 373 Apr 44

Exposure of red cells to fluoride produces a variety of metabolic alterations, most of which are based upon the secondary effects of enolase inhibition, which reduces pyruvate synthesis and interferes with the regeneration of diphosphopyridine nucleotide (NAD). Adenosine triphosphate (ATP) is consumed in the hexokinase and phosphofructokinase reactions but is not regenerated since the deficiency of NAD limits glyceraldehyde phosphate dehydrogenase. ATP depletion in the presence of fluoride and calcium induces a massive loss of cations and water. Of the other known sites of ATP utilization, membrane-bound ATPase is inhibited by fluoride, but the incorporation of fatty acids into membrane phospholipids is unaffected until ATP is depleted. The addition of methylene blue to fluoride-treated red cells regenerates NAD, permitting triose oxidation and the generation of 3-phosphoglycerate and 2,3-diphosphoglycerate. Enolase inhibition is then partially overcome by mass action, and sufficient glycolysis proceeds to maintain the concentration of ATP. This in turn prevents the massive cation and water loss, and permits membrane phospholipid renewal to proceed. Membrane ATPase activity is not restored by the oxidant so that normal cation leakage remains unopposed by cation pumping in red cells exposed to the combination of fluoride and methylene blue.
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PMID:Energy metabolism in human erythrocytes. I. Effects of sodium fluoride. 432 3

Inhibitor titration experiments carried out with carboxyatractyloside, oligomycin and rotenone show that in the case of heart mitochondria the membrane-bound ATPase and the respiratory chain are the major factors controlling the rate of oxidative phosphorylation whereas the adenine nucleotide carrier exhibits no control strength. As shown by carboxyatractyloside titration curves under different conditions, the relative importance of the adenine nucleotide carrier depends on the mode of regeneration (F1-ATPase or glucose plus hexokinase) of ADP from ATP exported outside mitochondria, on the total concentration of adenine nucleotides present in the medium and on the mode of limitation of the rate of respiration (cyanide, rotenone, oligomycin or mersalyl). Concomitantly with the inhibition of O2 consumption, carboxyatractyloside brings about a rise in membrane potential. The inverse relationship between the two processes is observed for carboxyatractyloside concentrations ranging between 0.7 and 1.5 nmol per mg protein. Carboxyatractyloside concentrations below and above this range increase the membrane potential without affecting significantly the rate of respiration. Titration experiments aimed at comparing the effects of ADP, carboxyatractyloside and the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, corroborate the conclusion that in heart mitochondria a major limiting factor in oxidative phosphorylation is the capacity of the respiratory chain.
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PMID:Control of oxidative phosphorylation in rat heart mitochondria. The role of the adenine nucleotide carrier. 608

The following aspects have been investigated in 10 patients affected by Huntington's disease )HD): --extensive haematological investigations; --red cell enzyme activities and level of the most important glycolytic intermediate compounds; --protein, lipid and carbohydrate composition of the erythrocyte membrane and membrane polarity; --effects of in vitro aging on red cell membranes. Lack of 4.5 protein band in SDS-PAGE and 14-fold decrease in membrane-bound catalase were found in the in vitro aged red cells from the 10 HD patients examined. Na+ + K+ATPase was slightly higher than normal in all the patients. Red cells from 5 out of 8 patients showed a decrease in reduced glutathione and phosphoenolpyruvate levels and/or an increase in hexokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase and glutathione reductase activities. The haematological investigations, the protein lipid and carbohydrate composition of the fresh red cells, the membrane polypeptide aggregates and the membrane polarity evaluated by microspectrofluorometric analysis were normal.
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PMID:Metabolic impairment and membrane abnormality in red cells from Huntington's disease. 644 71

The highly glucolytic hepatoma cell line H-91 is characterized by a high hexokinase activity to rat liver; 50% of this activity is associated with the mitochondrial fraction [Bustamante, E., & Pederson, P.L. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3735--3739]. Treatment of mitochondria from this cell line with adenosine 5'=triphosphate (ATP) or glucose 6-phosphate solubilizes bound hexokinase activity. Solubilization of the enzyme by ATP results in a six- to sevenfold purification. Free ATP, unchelated by Mg ions, induces the release of the enzyme from the membrane, whereas the MgATP complex is ineffective. Ethylenediaminetetraacetic acid (EDTA) fails to release mitochondrial hexokinase indicating that the enzyme is not attached to the membrane by divalent cations. Energization of mitochondria is not required for ATP to induce solubilization of bound hexokinase. This is evidenced by (a) the ability of the nonhydrolyzable ATP analogue adenylyl imidodiphosphate to solubilize the enzyme, (b) the inability of uncouplers and inhibitors of oxidative phosphorylation to either solubilize or prevent the release of mitochondrial hexokinase, and (c) the inability of atractyloside to solubilize or prevent the release of bound hexokinase. The bound and the ATP-solubilized forms of mitochondrial hexokinase from H-91 hepatoma cells are kinetically different. When membrane bound, the enzyme has a significantly higher apparent affinity (Km = 0.25 mM) for its substrate MgATP than when solubilized (Km = 1.2 mM). Free ATP acts as a competitive inhibitor of mitochondrial hexokinase. Both the membrane-bound and the solubilized forms of mitochondrial hexokinase have about the same apparent affinity for glucose (Km = 56 and 83 microM, respectively). The experiments reported here provide the first description of the properties and the nature of binding of mitochondrial hexokinase from a tumor cell line growing in tissue culture.
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PMID:Mitochondrial hexokinase of rat hepatoma cells in culture: solubilization and kinetic properties. 677 59

Conformational changes of hexokinase from ascites tumor cells have been studied by chemical modification of lysine residues with imidoesters with the following results: 1) The membrane-bound enzyme, in contrast to the soluble enzyme, is not inactivated by treatment with dimethyl suberimidate, which suggests (a) lysine residue(s) essential for the activity that is protected in the membrane-bound enzyme. 2) Three different conformations have been detected in the membrane-bound enzyme. Two of these are induced by glucose and glucose 6-phosphate, respectively. 3) Treatment of the membrane-bound enzyme with dimethyl suberimidate affects its sensitivity to the inhibition by glucose 6-phosphate, but not its activity or degree of maximal inhibition. This suggests that lysine(s) is related to the binding of glucose 6-phosphate to its allosteric regulatory site. 4) In intact tumor cells, most, if not all, of the hexokinase activity seems to be in a membrane-bound form.
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PMID:Mitochondrial membrane-bound hexokinase of ascites tumor cells. Functional implications of lysine residues studied by modification with imidoesters. 680 58

Macromolecules can restore the morphological changes in the outer mitochondrial compartment that occur upon isolation of the organelle. They decrease the volume of the intermembrane space and increase the number of intermembrane contact sites. In this study, we investigated the effects of macromolecules on one of the processes occurring in the mitochondrial outer compartment and for which the native structure might be important, i.e. the ADP supply from outer-membrane-bound hexokinase-I to oxidative phosphorylation. With the use of a reconstituted system in which rat liver mitochondria and extramitochondrial pyruvate kinase compete for ADP generated by hexokinase, it was shown that (a) part of the ADP generated by mitochondrially associated hexokinase is not accessible to pyruvate kinase and is channeled into the mitochondrion, (b) in the presence of 10% (mass/vol.) macromolecules (i.e. dextran M20 or BSA) the pyruvate kinase inaccessible fraction increases from 19% to 31% of the ADP produced by hexokinase, (c) the ADP channeling is a characteristic property of bound hexokinase, and (d) the increased channeling induced by macromolecules can neither be explained by direct effects of these macromolecules on the basic respiratory properties of rat liver mitochondria, nor by direct effects of the kinetic properties of hexokinase-I. ATP and ADP determinations were performed in hexokinase/mitochondria incubation mixtures in the presence of macromolecules. These determinations showed that an important consequence of the channeling capacity of bound hexokinase is that lower extramitochondrial ADP levels and consequently higher extramitochondrial ATP/ADP ratios are maintained than when hexokinase is not bound. The experimental data demonstrate that the ADP channeling activity associated with bound hexokinase leads to the formation of two ADP concentration gradients, one across the outer membrane and one between bound hexokinase and the bulk phase.
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PMID:Macromolecules increase the channeling of ADP from externally associated hexokinase to the matrix of mitochondria. 755 9

After tissue homogenization, 43% of the total hexokinase activity found in maize radicles was recovered in the mitochondrial fraction and 35% was soluble, in the cytosol. The maize submitochondrial particles obtained after mitochondrial sonication retained a high hexokinase activity. The mitochondrial respiration (state 4 rate) was activated by glucose. This activation was blocked by carboxyatractyloside (0.5 mM) and by oligomycin (2 micrograms/ml). The affinities for ATP and glucose of both soluble and membrane-bound maize hexokinases are similar to those of yeast hexokinase. The Km for ATP of these different forms of hexokinase varied between 0.15 and 0.37 mM, and the Km for glucose between 0.05 and 0.13 mM. A major difference between the two maize hexokinase forms is that only the mitochondrial enzyme was strongly inhibited by ADP (Ki 0.04 mM). The soluble forms of hexokinase found both in the cytosol of maize radicles and in yeast are not inhibited by ADP. In a previous report [de Meis, Grieco and Galina (1992) FEBS Lett. 308, 197-201] it was shown that the mitochondrial F1-F0-ATPase can use glucose 6-phosphate and yeast hexokinase as an ATP regenerating system. We now show that the membrane-bound hexokinase and glucose 6-phosphate can also serve as an ATP regenerating system for the mitochondria of maize radicles provided that the ADP concentration is kept below 0.05 mM. Higher ADP concentrations inhibit the reverse reaction of the mitochondrial hexokinase.
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PMID:Different properties of the mitochondrial and cytosolic hexokinases in maize roots. 761 43

The activity, intracellular distribution and mRNA expression of hexokinase isoenzymes were studied in normal rat liver, and in epithelial liver cells at different stages of neoplastic transformation, including non-tumorigenic and tumorigenic cell lines. In contrast to liver, all transformed cells exhibited only hexokinase I and II, which both showed significantly increased activity, hexokinase II being the more abundant form. In parallel, the mRNA expression of the two isoenzymes was elevated, indicating transcriptional control of gene expression. Hexokinase I and II were found in the cytosol and bound to mitochondrial membranes; the percentage of membrane-bound enzyme activity increased with the grade of transformation from 32% of total activity in normal liver up to 69% in dedifferentiated tumor cells. The ratio of hexokinase I/II was higher in the membrane fraction than in the cytosol. In all tissues studied hexokinase II could be resolved in two subtypes IIa and IIb by hydrophobic interaction chromatography. The relative proportion of cytosolic IIa and IIb varied significantly between normal liver (1:1) and transformed cells, and among cells of different transformation stages (4:1 to 1:10). IIa demonstrated the main activity in the more differentiated, IIb in the less differentiated cell lines. IIa-activity showed a good correlation with the intracellular glucose 6-phosphate concentration of the cells. The data indicate that neoplastic cell transformation is accompanied by progressive alterations in the proportion and subcellular distribution of hexokinase isoenzymes I and II.
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PMID:Differences in expression and intracellular distribution of hexokinase isoenzymes in rat liver cells of different transformation stages. 794 23


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