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)

A practical biosensor system has been developed for the determination of urinary glucose using a flow-injection analysis (FIA) amperometric detector and ion-exchange chromatography. Glucose oxidase was immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an immobilized enzyme column. On the basis of its negative charge at pH 5.5, endogenous urate in urine samples was effectively retained by an upstream anion-exchange resin column. The biosensor system possessed a sensitivity of 160 +/- 2.4 RU microM-1 (RU or relative unit is defined as 2.86 microV at the detection output) for glucose with a minimum detection level of 10 microM. When applied for the determination of urinary glucose, the result obtained compared very well with that of the widely accepted hexokinase assay. The immobilized glucose oxidase could be reused for more than 1000 repeated analyses without losing its original activity. The reuse of the acetate anion-exchange column before replacement would be about 25-30 analyses. Acetaminophen and ascorbic acid were also effectively adsorbed by the acetate anion exchanger. The introduction of this type of anion exchanger thus greatly improved the selectivity of the FIA biosensor system and fostered its applicability for the determination of glucose in urine samples.
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PMID:Determination of urinary glucose by a flow injection analysis amperometric biosensor and ion-exchange chromatography. 130 63

In this paper we report the purification of pig erythrocyte hexokinase type III, at preparative level, using 52 liters of starting material (hemolysate). This was possible using a new efficient anion exchanger support, the Toyopearl DEAE 650 M which allows completely to change the strategy of removing hemoglobin from hemolysates, permitting to handle large amounts of starting material and reducing work would have required months using conventional anion exchanger supports, to only 2-3 days. Furthermore, we have tested the binding of other red blood cell enzymes to the Toyopearl DEAE 650 M, showing a wider potential use of this chromatographic support for their purification at a preparative level.
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PMID:Preparative purification of pig red blood cell hexokinase type III using a new efficient chromatographic support. 162 Jun 86

A chemiluminescence fiber optic system coupled to flow injection analysis (FIA) and ion exchange chromatography has been developed for determining glucose in blood and urine. Immobilized glucose oxidase acted on beta-D-glucose to produce hydrogen peroxide, which was then reacted with luminol in the presence of ferricyanide to produce a light signal. Endogenous ascorbic acid and uric acid present in urine or blood samples were effectively retained by an upstream acetate anion exchanger. In addition, acetaminophen could also be adsorbed by this ion exchanger. The detection system exhibited a sensitivity of 1.315 +/- 0.044 RU microM-1 for glucose with a minimum detection level of 1 microM. When applied for the determination of urinary and blood glucose levels, the results obtained compared well with those of the reference hexokinase assay. Immobilized glucose oxidase was reused for over 500 analyses without losing its original activity. A conservative estimate for the reuse of the acetate ion exchange column was about 100 analyses.
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PMID:On-line chemiluminescence assay using FIA and fiber optics for urinary and blood glucose. 776 30

A flow injection analysis (FIA) biosensor system has been developed for the determination of glucose from urine, blood plasma and foodstuffs. Glucose oxidase was immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an enzyme column. The hydrogen peroxide released from the conversion of glucose to gluconic acid was monitored by a platinum electrode vs. silver/silver chloride poised at +700 mV. As a novel aspect to the improvement of the selectivity of the biosensor system, an anion exchange column was placed upstream to remove uric acid, ascorbic acid or acetaminophen, three major electroactive interfering substances which usually occur in urine and blood plasma. Among several resins tested, the effective adsorption of uric and ascorbic acids could be accomplished using an acetate anion exchanger, and the selectivity coefficient was pH dependent. The binding of acetaminophen to the resin was much less efficient and, in all cases, the selectivity coefficient was independent of the operating temperature up to 37 degrees C. When applied to real samples, the data obtained by the biosensor system compared well with those of the standard hexokinase assay. The immobilized glucose oxidase could be reused for at least 2000 repeated analyses without loss of its original activity.
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PMID:Improvement of the selectivity of an FIA amperometric biosensor system for glucose. 839 49

In vitro incubation of isolated hexokinase isozyme I or isolated dimer of mitochondrial creatine kinase with the outer mitochondrial membrane pore led to high molecular weight complexes of enzyme oligomers. Similar complexes of hexokinase and mitochondrial creatine kinase could be extracted by 0.5% Triton X-100 from homogenates of rat brain. Hexokinase and creatine kinase complexes could be separated by subsequent chromatography on DEAE anion exchanger. The molecular weight, as determined by gel-permeation chromatography, was approximately 400 kDa for both complexes. The Mr suggested tetramers of hexokinase (monomer 100 kDa) and creatine kinase (active enzyme is a dimer of 80 kDa). The composition of the complexes was further characterised by specific antibodies. Besides either hexokinase or creatine kinase molecules the complexes contained porin and adenylate translocator. It was possible to incorporate the complexes into artificial bilayer membranes and to measure conductance in 1 M KCI. The incorporating channels had a high conductance of 6 nS that was asymmetrically voltage dependent. The complexes were also reconstituted in phospholipid vesicles that were loaded with ATP. Complex containing vesicles retained ATP while vesicles reconstituted with pure porin were leaky. The internal ATP could be used by creatine kinase and hexokinase in the complex to phosphorylate external creatine or glucose. This process was inhibited by atractyloside. The hexokinase complex containing vesicles were furthermore loaded with malate or ATP that was gradually released by addition of Ca2+ between 100 and 600 microM. The liberation of malate or ATP by Ca2+ could be inhibited by N-methylVal-4-cyclosporin, suggesting that the porin translocator complex constitutes the permeability transition pore. The results show the physiological existence of kinase porin translocator complexes at the mitochondrial surface. It is assumed that such complexes between inner and outer membrane components are the molecular basis of contact sites observed by electron microscopy. Kinase complex formation may serve three regulatory functions, firstly regulation of the kinase activity, secondly stimulation of oxidative phosphorylation and thirdly regulation of the permeability transition pore.
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PMID:Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore. 891 85

Complexes between hexokinase, outer membrane porin, and the adenylate translocator (ANT) were recently found to establish properties of the mitochondrial permeability transition pore in a reconstituted system. The complex was extracted by 0.5% Triton X-100 from rat brain membranes and separated by anion exchanger chromatography. The molecular weight was approximately 400 kDa suggesting tetramers of hexokinase (monomer 100kDa). By the same method a porin, creatine kinase octamer, ANT complex was isolated and reconstituted in liposomes. Vesicles containing the reconstituted complexes both retained ATP that could be used by either kinase to phosphorylate external creatine or glucose. Atractyloside inhibited this activity indicating that the ANT was involved in this process and was functionally reconstituted. Exclusively from the hexokinase complex containing liposome internal malate or ATP was released by addition of Ca2+ in a N-methylVal-4-cyclosporin sensitive way, suggesting that the hexokinase porin ANT complex might include the permeability transition pore (PTP). The Ca2+ dependent opening of the PTP-like structure was inhibited by ADP (apparent I(50), 8 microM) and ATP (apparent I(50), 84 microM). Also glucose inhibited the PTP-like activity, while glucose-6-phosphate abolished this effect. Although porin and ANT were functionally active in vesicles containing the creatine kinase octamer complex, Ca2+ did not induce a release of internal substrates. However, after dissociation of the creatine kinase octamer, the complex exhibited PTP-like properties and the vesicles liberated internal metabolites upon addition of Ca2+. The latter process was also inhibited by N-methylVal-4-cyclosporin. The activity of peptidyl-prolyl-cis-trans-isomerase (representing cyclophilin) was followed during complex isolation. Cyp D was co-purified with the hexokinase complex, while it was absent in the creatine kinase complex. The inhibitory effect of N-methylVal-4-cyclosporin on the creatine kinase complex may be explained by direct interaction with the creatine kinase dimer that appeared to support octamer formation.
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PMID:Complexes between porin, hexokinase, mitochondrial creatine kinase and adenylate translocator display properties of the permeability transition pore. Implication for regulation of permeability transition by the kinases. 945 79