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)

Activity of hexokinase, phosphorylase, glucoso-6-phosphate dehydrogenase lactate-dehydrogenase was studied in liver slices, homogenate and supernatant fraction after freezing at a rate of 1 degree/min down to -30 degrees C. The enzyme activity in homogenate and supernatant fraction does not change after freezing. A significant reduction in the activity of most enzymes that is followed by an increase in their activity in the freezing medium was observed in the experiments. Cryoprotectant polyethylene glycol, mol. wt. 300 and 1,000 (PEG-300 and PEG-1,000), partially prevents the observed changes in the enzyme activity; PEG-1,000 is more effective than PEG-300. Experimental results show that the main reason for the reduction of the enzyme activity observed after freezing the tissue slices is a decrease in the volume of intracellular enzyme proteins due to their leakage from the injured cellular elements into the exocellular medium.
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PMID:[Activity of carbohydratephosphate metabolism enzymes in liver slices after freezing and thawing]. 88 19

Human and rat erythrocytes were fractionated by counter-current distribution in charge-sensitive dextran/poly(ethylene glycol) two-phase systems. The specific activities of the key glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) declined along the distribution profiles, although the relative positions of the activity profiles were reversed in the two species. These enzymes maintained their normal response to specific regulatory effectors in all cell fractions. No variations were observed for phosphoglycerate kinase and bisphosphoglycerate mutase activities. Some correlations between enzyme activities (pyruvate kinase/hexokinase, pyruvate kinase/phosphofructokinase, pyruvate kinase/pyruvate kinase plus phosphoglycerate kinase, pyruvate kinase/bisphosphoglycerate mutase and phosphoglycerate kinase/bisphosphoglycerate mutase ratios) were studied in whole erythrocyte populations as well as in cell fractions. These results strongly support the fractionation of human erythrocytes according to cell age, as occurs with rat erythrocytes.
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PMID:Changes in glycolytic enzyme activities in aging erythrocytes fractionated by counter-current distribution in aqueous polymer two-phase systems. 165 39

The utilization of isocratic, reverse-phase, ion-paired high-performance liquid chromatography for analysis of creatine phosphate allows for rapid quantification of multiple samples. Cryogenic sample handling and the addition of ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid as a Ca2+ sequestering agent during perchloric acid extraction enhance maximal recovery of creatine phosphate from brain samples. Peak identification is supported by a complete enzymatic shift with a phosphocreatine kinase, hexokinase, and glucose-6-phosphate dehydrogenase system.
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PMID:Determination of creatine phosphate levels in brain tissue by isocratic reverse-phase, ion-paired high-performance liquid chromatography. 673 48

Liquid-liquid partition chromatography in an aqueous poly(ethylene glycol)/dextran two-phase system (LLPC) is shown to be a quick and sensitive method for detecting conformational changes occurring upon binding of ligands by biospecific molecules. Two groups of well-characterized proteins, enzymes and monoclonal antibodies, were employed. As an example, LLPC demonstrated that isoforms of lactate dehydrogenase as well as of hexokinase existed in a ligand-dependent equilibrium between two forms and that conformational changes occurred when monoclonal antibodies bound haptens. We also demonstrate that the method could be used to detect and separate subfractions in preparations of unliganded proteins that appeared to be homogeneous when analysed by other techniques.
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PMID:A new approach to examine conformational changes occurring upon binding of ligand by biomolecules. 753 94

Physiological increases in matrix calcium are known to stimulate three mitochondrial dehydrogenases. In mitochondria isolated from rat heart, calcium stimulates rates of State 3 respiration during oxidation of succinate and of several NAD-linked substrates. In this study, we investigated the effects of calcium on NADH dehydrogenase and succinate dehydrogenase activities since the mechanism of these effects is unresolved. The respiratory activities of intact mitochondria and submitochondrial particles (SMP) were compared during incubation in media containing either ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) or a Ca2+/EGTA buffer (approximately 1 microM free Ca2+). In intact mitochondria oxidizing 20 mM glutamate plus 2 mM malate, the membrane potential (delta psi) and matrix NAD(P)H were maintained at higher levels, and the maximal rate of ADP-stimulated respiration (State 3) was increased twofold by the presence of calcium. With succinate as substrate, calcium stimulated State 3 respiration but it did not influence the pyridine nucleotides redox state or membrane potential. Stimulation of succinate-supported respiration by addition of 6-10 microM ADP in the presence of hexokinase caused a sudden decrease in NAD(P)H and collapse of delta psi. This effect was not caused by inhibition of succinate dehydrogenase or by opening of the nonspecific pore. Calcium did not influence the oxidation of succinate by SMP containing either activated or nonactivated succinate dehydrogenase. In addition, calcium did not alter the kinetics of succinate dehydrogenase activation. Calcium and magnesium, in the concentration range of 0.02 to 5 mM, did not influence the NADH dehydrogenase activity of SMP. Energization of SMP by oligomycin addition, however, dramatically influenced the kinetic properties of NADH dehydrogenase. It is proposed that in heart mitochondria, calcium does not affect directly the components of electron transport but it may influence the activity of NADH dehydrogenase indirectly by increasing delta psi.
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PMID:Influence of calcium on NADH and succinate oxidation by rat heart submitochondrial particles. 786 38

Yeast hexokinase, a homodimer (100 kDa), is an important enzyme in the glycolytic pathway. Although Cibacron Blue 3G-A (Reactive Blue 2) has been previously shown to inactivate yeast hexokinase, no comprehensive study exists concerning the nature of interaction(s) between hexokinase and the blue dye. A comparison of the computer-generated three-dimensional (3D) representations showed considerable overlap of the purine ring of ATP, a nucleotide substrate of hexokinase, with the hydrophobic anthraquinone moiety of the blue dye. The visible spectrum of the blue dye showed a characteristic absorption band centred at 628 nm. The visible difference spectrum of increasing concentration of the dye and the same concentrations of the dye plus a fixed concentration of hexokinase exhibited a maximum, a minimum and an isobestic point at 683, 585, and 655 nm respectively. The visible difference spectrum of the blue dye and the dye in 50% ethylene glycol showed a maximum and a minimum at 660 and 570 nm respectively. The visible difference spectrum of the blue dye in the presence of the dye and hexokinase modified at the active site by pyridoxal phosphate, iodoacetamide and o-phthalaldehyde was devoid of bands characteristic of the hexokinase-blue dye complex. Size-exclusion-chromatographic studies in the absence or presence of guanidinium chloride showed that the enzyme inactivated by the blue dye was co-eluted with the unmodified enzyme. The dialysis residue obtained after extensive dialysis of the gel-filtered complex, against a buffer of high ionic strength, showed an absorption maximum at 655 nm characteristic of the dye-enzyme complex. Inactivation data when analysed by 'Kitz-Wilson'-type kinetics for an irreversible inhibitor, yielded values of 0.05 min-1 and 92 microM for maximum rate of inactivation (k3) and dissociation constant (Kd) for the enzyme-dye complex respectively. Sugar and nucleotide substrates protected hexokinase against inactivation by the blue dye. About 2 mol of the blue dye bound per mol of hexokinase after complete inactivation. The inactivated enzyme could not be re-activated in the presence of 1 M NaCl. These results suggest that Cibacron Blue 3G-A inactivated hexokinase by an irreversible adduct formation at or near the active-site. Spectral and kinetic studies coupled with an analysis of the 3D representations of model compounds corresponding to the substructures of the blue dye suggest that 1-amino-4-(N-phenylamino)anthraquinone-2-sulphonic acid part of the blue dye may represent the minimum structure of Cibacron Blue 3G-A necessary to bind hexokinase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inactivation of yeast hexokinase by Cibacron Blue 3G-A: spectral, kinetic and structural investigations. 819 58

Procion or Cibacron blue dyes, containing polynuclear aromatic rings and mono- and dichlorotriazine nuclei, immobilized on dextran matrices, have been used for over a decade to purify diverse groups of enzymes by dye-ligand chromatography. Comparatively less attention has been paid to investigating the nature of molecular interactions between similarly constituted red dyes and various enzymes so as to ascertain their potential and thus justify their use in the purification of enzymes by dye-ligand chromatography. We investigated and found that Cibacron brilliant red 3B-A, a monochlorotriazine dye, inhibited phosphotransferase activity of yeast hexokinase. The dissociation constant, KD, and the rate of dye-enzyme complex formation, k3, were 120 microM and 0.1 min-1, respectively. The enzyme was protected from inactivation by sugar and nucleotide substrates. About 2 mol of the dye bound per mole of the enzyme. The chromophore of the dye showed absorption at 524 nm. The visible difference spectrum of increasing concentration of the dye and same concentrations of the dye plus a fixed concentration of hexokinase exhibited a maximum, a minimum, and an isosbestic point at 569, 501, and 512 nm, respectively. The difference spectrum of the dye and dye in 60% ethylene glycol showed a maximum and a minimum at 556 and 495 nm, respectively. The dye showed no visible difference spectrum in the presence of hexokinase modified at the active site by iodoacetamide, pyridoxal phosphate, and o-phthalaldehyde. Hexokinase modified by the dye coeluted with the unmodified enzyme during size-exclusion chromatography in the absence or presence of guanidinium hydrochloride. These results suggest that the dye interacts with the hydrophobic environment of the active site of the enzyme. Analysis of the kinetics of inhibition of hexokinase by model compounds and comparison of their computer-assisted three-dimensional representations with that of Cibacron brilliant red 3B-A suggest that 1-amino-8-naphthol-3,6-disulfonic acid may represent the minimum structure for the dye to bind.
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PMID:Inactivation of yeast hexokinase by Cibacron brilliant red 3B-A. 851 15

Evidence is presented that intermediates of the oxidative pentose phosphate pathway (OPPP) are channeled from one pathway enzyme to the next. CO2 produced from [1-14C]glucose in the presence of unlabelled pathway intermediates contained much more radioactivity than predicted by a model in which pathway-produced intermediates are in equilibrium with identical molecules in the bulk phase. This was the case whether glucose 6-phosphate (Glc6P), 6-phosphogluconolactone, or 6-phosphogluconate was added. Assumptions involved in calculating the amount of 14CO2 predicted for free mixing of 14C-labelled and unlabelled intermediates are discussed, together with the following results. (a) 14CO2 production by pea nodules in the presence of 3 mM 6-phosphogluconate was higher than in its absence. (b) Apparent channeling of intermediates was much higher for purified yeast enzymes than for yeast extract. (c) 6-Phosphogluconate and 6-phosphogluconolactone were channeled between yeast Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase despite the absence of 6-phosphogluconolactonase in the purified yeast enzyme mixture. (d) When purified yeast hexokinase was physically separated from Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase by a dialysis membrane, there was no apparent channeling. (e) Poly(ethylene glycol), high salt and detergents had little effect on apparent channeling of OPPP intermediates, which is consistent with a stable complex of enzymes. On the other hand, density gradient centrifugation experiments suggested a more transient interaction between the enzymes. Taken together, the results support channeling of OPPP pathway intermediates.
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PMID:Evidence for channeling of intermediates in the oxidative pentose phosphate pathway by soybean and pea nodule extracts, yeast extracts, and purified yeast enzymes. 920 16

This paper describes the preparation of oligo(ethylene glycol)-terminated alkyltrichlorosilanes, Cl3Si(CH2)11(OCH2CH2)(n)OCH3 (n = 2, 3), and their use in the formation of self-assembled monolayers on an oxide surface. The adsorption of the trichlorosilanes from solution produces densely packed, oriented monolayer films that are 2-3 nm in thickness. The trichlorosilyl group anchors the molecules to the surface, and the resulting film exposes the ethylene glycol units at its surface, as noted by its moderate hydrophilicity (theta2(H2O) approximately 68 degrees). The films are robust with stabilities similar to those of other alkylsiloxane coatings. These oligo(ethylene glycol)-terminated silane reagents produce films that notably exhibit resistances against the non-specific adsorption of proteins from solution that are better than for films prepared from octadecyltrichlorosilane. With insulin, tysozyme, albumin, and hexokinase, no adsorption was observed with the oligo(ethylene glycol)-siloxane coatings whereas protein films of approximately a monolayer formed on surfaces-treated with octadecyltrichlorosilane. With fibrinogen, complete resistance was not possible with either coating; however, the oligo(ethylene glycol)-siloxane coatings exhibited greater resistance against non-specific adsorption. The oligo(ethylene glycol)-siloxane coatings offer performance advantages over available systems and could easily provide a direct and superior replacement in protocols that presently use silane reagents to generate hydrophobic, 'inert' surfaces.
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PMID:Protein-resistant coatings for glass and metal oxide surfaces derived from oligo(ethylene glycol)-terminated alkytrichlorosilanes. 984 2

We have previously reported that sucrose modulates anthocyanin biosynthesis in cell suspension cultures of Vitis vinifera L. The main role of sugar in this response does not seem to be that of general carbohydrate source for the supply of energy. In the present work, a number of pharmacological agents were used to further investigate the components of the signal transduction pathway involved in the induction of anthocyanin biosynthesis by sugar. We found that the phosphorylation of hexose by hexokinase, but not its transport, has to be taken into account for the sucrose signal transduction leading to anthocyanin accumulation. Indeed, 3-O-methylglucose, a glucose analog transported into cells but not phosphorylated by hexokinase, has no effect on anthocyanin production. Mannose mimics the effect of sucrose in grape cells, and mannoheptulose, a specific inhibitor of hexokinase, reduces the accumulation of anthocyanins in response to sucrose. The results with the two latter analogs are discussed. Ca2+ channel blockers, verapamil and LaCl3, which were used to investigate the role of extracellular Ca2+, all inhibited the sugar response. Ca2+ depletion by pretreatment with ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) also blocked the sugar response, which was partially recovered when Ca2+ was added exogenously after Ca2+ depletion. The use of two potent calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphtalenesulphonamide (W7) and chlorpromazine, showed that calmodulin is involved in the sugar signal transduction. A protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), and the protein phosphatase inhibitors, endothall and cantharidin, also inhibited the sugar response. The results of the present study suggest the involvement of several components of general signal transduction pathways such as Ca2+, calmodulin, and protein kinases phosphatases in the induction of anthocyanin biosynthesis by sugar.
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PMID:Sugar sensing and Ca2+-calmodulin requirement in Vitis vinifera cells producing anthocyanins. 1074 78


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