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 mitochondrial hexokinase from rat brain, selectively released from mitochondria by the action of glucose 6-phosphate, can be purified to greater than 90% homogeneity by a single affinity chromatography step on Affi-Gel Blue; the Cibacron Blue F3GA ligand bound to this matrix serves as an analog of ATP, the normal substrate for the enzyme, and selective elution is accomplished using glucose 6-phosphate which is a competitive ligand vs. ATP. With this and other modifications to the previously described procedure highly purified enzyme is readily obtained in good yield and with retention of the ability to rebind to mitochondria.
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PMID:Rapid purification of mitochondrial hexokinase from rat brain by a single affinity chromatography step on Affi-Gel blue. 274 Feb 89

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) prepared from baker's yeast binds to immobilized Cibacron Blue F3G-A and Procion Red HE-3B. In this paper the two dyes are compared with respect to their use in the purification of this enzyme. Cibacron Blue chromatography was found useful at an early stage of purification for the removal of contaminating hexokinase, phosphoglucose isomerase and phosphoglucomutase. With Procion Red HE-3B Sepharose the NADP dependent enzymes phosphogluconate dehydrogenase and glutathione reductase are separable from glucose-6-phosphate dehydrogenase. Unlike Cibacron Blue gel chromatography, the enzyme can be specifically eluted from Procion Red HE-3B Sepharose by a NADP gradient. Other monochlorotriazine dyes like Xirone Brillant Red BHD, 4BHD, 6BHD and GHD and the dichlorotriazine dye Procion Brown MX-5BR immobilized to Sepharose have only little binding affinity to glucose-6-phosphate dehydrogenase. The binding behaviour of different immobilized triazine dyes for pre-purified and purified glucose-6-phosphate dehydrogenase is compared. In addition, the influence of the free dyes on the activity of glucose-6-phosphate dehydrogenase is studied. It is demonstrated that the results of kinetic and binding studies with the purified enzyme are not uncritically applicable for the selection of a dye as ligand for affinity chromatography during enzyme preparation.
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PMID:Interactions of immobilized and free triazine dyes with glucose-6-phosphate dehydrogenase from yeast. 351 9

Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1) is inhibited by acidic phospholipids such as phosphatidylinositol, phosphatidylserine, and cardiolipin. Several aspects of this inhibition are atypical when compared to inhibition by established reversible inhibitors of this enzyme such as the product, Glc-6-P. Maximal inhibition is attained rather slowly (approximately 30 min at 22 degrees C), and is not reversed by simple dilution of the enzyme-lipid mixture. Ligands such as ATP or Glc-6-P can protect the enzyme against inhibition by acidic phospholipids; addition of protective ligands after mixing of enzyme and lipids does not, however, reverse inhibition that occurred prior to ligand addition. Inhibition can be prevented but not reversed by elevated (0.1-0.2 M) [NaCl], indicating a probable role for electrostatic forces in the interaction of lipid with enzyme. Greater inhibition is seen at 22 degrees C than at 3-4 degrees C, suggesting that hydrophobic interactions may also be involved. It is suggested that acidic phospholipids inhibit brain hexokinase by binding at the nucleotide-binding site of the enzyme. The effectiveness of ATP (or the ATP analog, Cibacron Blue) in protecting against inhibition by acidic phospholipids is attributed to direct competition between ATP and the phospholipid for a common binding site. The effectiveness of Glc-6-P (or analogs) in preventing the inhibition is attributed to a conformational change, induced by the binding of this ligand, which prevents binding of ATP or acidic phospholipids to the enzyme. The pH dependency of the inhibition has suggested involvement of the protonated form of a dissociable group (pK approximately 7) on the enzyme in the interaction with acidic phospholipids; this may be the histidyl residue implicated by Solheim and Fromm [Biochemistry 19, 6074-6080 (1984)] in the binding of ATP to brain hexokinase. Structural similarities in the nucleotide-binding sites of several nucleotide-binding enzymes suggest that similar inhibition by acidic phospholipids may be seen with other enzymes of this type; there are already some reports to this effect.
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PMID:Acidic phospholipids may inhibit rat brain hexokinase by interaction at the nucleotide binding site. 396 91

Bivalent metal ions, particularly Zn2+ and other members of the first-row transition series, promote irreversible inactivation of yeast hexokinase by Cibacron Blue F3G-A at a site competitive with both ATP and D-glucose. Difference spectroscopy indicates that the protein-dye dissociation constant is decreased from 250 micrometers in the absence of metal ions to less than 100 micrometers in the presence of appropriate concentrations of metal ions, with specificity displayed in the sequence of Zn2+ greater than Cu2+ greater than Ni2+ greater than Mn2+. Quantitative inactivation of yeast hexokinase leads to the incorporation of approx. 1 mol of Cibacron Blue F3G-A/mol of subunit of mol. wt. 51 000 in both the presence and the absence of metal ion. These results suggest the formation of a highly specific ternary complex involving enzyme, dye and metal ion at the active-site region of the enzyme, and correlate well with the known effects of metal ions in promoting the binding of hexokinase to immobilized Cibacron Blue F3G-A.
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PMID:Metal-ion-promoted binding of triazine dyes to proteins. The interaction of Cibacron Blue F3G-A with yeast hexokinase. 675 35

Low concentrations of metal ions, particularly those of the first row transition series such as Zn2+, Co2+, Mn2+, Ni2+, Cu2+, and, to a lesser extent, the group IIA ions, Ca2+ and Mg2+, promotes binding of carboxypeptidase G2, alkaline phosphatase and yeast hexokinase to immobilized Procion Red H-8BN, Procion Yellow H-A and Cibacron Blue F3G-A respectively. The binding of ovalbumin to immobilized Cibacron Blue F3G-A and Procion Orange MX-G is selectively enhanced in the presence of AI3+. With ovalbumin and alkaline phosphatase, the effect is almost totally specific for both the metal ion and dye, whereas with carboxypeptidase G2 and hexokinase, metal ions such as Co2+, Ni2+, Mn2+, Cu2+, Ca2+ and Mg2+ also promote binding to varying degrees. Almost all other monovalent and trivalent metal ions appear to be ineffective. Metal ion-bound enzymes can subsequently be eluted with appropriate chelating agents of the amine, aminocarboxylate or substituted pyridine classes.
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PMID:Metal ion-promoted binding of proteins to immobilized triazine dye affinity adsorbents. 689 1

A number of reactive dichlorotriazine dyes specifically and irreversibly inactivate pig heart lactate dehydrogenase, yeast glucose 6-phosphate dehydrogenase and yeast hexokinase at sites competitive with NAD+, NADP+, and ATP respectively. Monochlorotriazine dyes, including Cibacron Blue F3G-A, do not inactivate lactate dehydrogenase but display high affinity and thus inhibit the inactivation by dichlorotriazine dyes. These data are interpreted in terms of the ability of nucleotide-binding enzymes to bind polysulphonated aromatic chromophores.
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PMID:Triazine dyes, a new class of affinity labels for nucleotide-dependent enzymes. 700 86

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

High hexokinase activity was not related to glucose repression in Candida utilis IGC 3092. The addition of Cibacron Blue 3G-A to growing cells in batch culture led to a permanent in vivo hexokinase inactivation, decreased growth rate and inhibited alcohol dehydrogenase. Hexokinase inactivation up to 90% did not alleviate glucose repression of alpha-glucosidase, as has been described for Saccharomyces cerevisiae and other yeasts. Moreover, when cells were physiologically derepressed by growing them in a chemostat at low glucose concentrations, the highest hexokinase activity was shown by the derepressed cells, and decreased as repression increased. Thus, in our strain of C. utilis, hexokinase activity was inversely proportional to glucose repression.
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PMID:The inactivation of hexokinase activity does not prevent glucose repression in Candida utilis. 859 74

The partitioning of glucose-6-phosphate dehydrogenase (G6PDH) (E.C. 1.1.1.49) and hexokinase (E.C. 2.7.1.1) in polyethylene glycol (PEG)-hydroxypropyl starch (PES) and PEG-phosphate aqueous two-phase systems was investigated with free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, as their affinity ligands. It was found that the free reactive triazine dyes, not bound to phase-forming polymers, preferentially partitioned in the top-PEG phase in the PEG-salt and PEG-PES systems. The effect of various parameters such as type and concentration of affinity ligands, pH of the system, molecular mass of PEG and phase composition on partitioning of the enzymes was estimated. Phosphate is a key factor affecting the enzyme partitioning in the PEG-PES system. Cibacron F3GA changed the partition coefficient of G6PDH from 0.73 to 1.59.
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PMID:Affinity partitioning of glucose-6-phosphate dehydrogenase and hexokinase in aqueous two-phase systems with free triazine dye ligands. 1238 80

To improve the selectivity of glucose-6-phosphate dehydrogenase (G6PDH) extraction by an aqueous two-phase system, a simple and inexpensive affinity aqueous two-phase system using unbound reactive triazine dyes as ligands was introduced. In a polyethylene glycol (PEG)/hydroxypropyl starch (PES) system, the unbound free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, partitioned unevenly in the top PEG-rich phase and thus showed an affinity effect on G6PDH, but no influence on hexokinase. The various parameters investigated were pH of the system, buffers, molecular weight of PEG, and ligand type and concentration. A two-step affinity extraction process was established for the purification of G6PDH from baker's yeast. The total yield of G6PDH was 66.9% and purification factor was 2.35.
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PMID:Purification of glucose-6-phosphate dehydrogenase from baker's yeast in aqueous two-phase systems with free triazine dyes as affinity ligands. 1272 23


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