Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Bacterial luciferase and NADH:FMN oxidoreductase have been immobilized onto arylamine glass beads. These immobilized enzymes can detect as little as 0.2 pmol of NADH per assay sample. Glucose-6-phosphate dehydrogenase has been co-immobilized with these enzymes, and with this system it is possible to quantitate 1 pmol of glucose 6-phosphate. By co-immobilizing a fourth enzyme, hexokinase, onto the glass beads, the system can reproducibly detect 20 pmol of glucose per liter. These immobilized enzyme systems are potentially superior to soluble enzymes by being reusable and much more stable. We compared the light-emitting properties of the immobilized enzyme systems with that of an equivalent mixture of the soluble enzymes. The most striking difference was the apparently more efficient conversion of NADH or glucose 6-phosphate to light by the immobilized enzymes. We used hydroxysteroid dehydrogenase in developing a soluble coupled system for the assay of androsterone and testosterone. The lower limit of detection was 100 pmol.
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PMID:Properties and uses of immobilized light-emitting enzyme systems from Beneckea harveyi. 3 21

Bioluminescent methods are widely used for the assay of the co-factors, NADH and ATP. Although the bioluminescent method is highly sensitive, the enzymes used are unstable and expensive. Therefore a chemiluminescent method would be valuable in clinical routine assay. We have developed a chemiluminescent method for the assay of NADH using the 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperoxidase(m-POD) system. In order to increase the sensitivity of this method, enzymatic cycling system was coupled to the chemiluminescent assay of NADH. Alcohol dehydrogenase and malate dehydrogenase were used as the cycling enzyme. The standard curve was obtained in the range from 3 X 10(-14) to 5 X 10(-12) mol/assay. The detection limit of NADH was 30 fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase. Two chemiluminescent methods for the assay of ATP have been developed. Method 1 is the system using hexokinase/G6PDH and 1-PMS/IL/m-POD, and method 2 is the system based on the enzymatic cycling reaction of ATP using hexokinase/pyruvate kinase. Method 2 is 1000/fold more sensitive than the method 1. The detection limit of ATP was 10 fmol/assay.
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PMID:Chemiluminescent assay of co-factors. 280 Dec 32

AMP is converted to ATP by incubating overnight with pyruvate kinase, phosphoenolpyruvate and adenylate kinase in the presence of endogenous ATP (ADP) as primer. In a subsequent incubation in the presence of pyruvate kinase, phosphoenolpyruvate, radioactive glucose and hexokinase, ATP and ADP are estimated together by coupling their recycling to the formation of glucose 6-phosphate. The latter is separated by precipitation using 76% (v/v) acetone for radioactivity measurement in the same Eppendorf tube. The sensitivity of these simple procedures matches or exceeds those of luciferase methods of nucleotide determination.
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PMID:Radioisotopic assay of femtomole quantities of total adenine nucleotides, ATP plus ADP, and AMP. 673 60

We have previously reported that cis-unsaturated free fatty acids, e.g. linoleic acid, introduced as free fatty acids into lymphocyte membranes inhibited surface immunoglobulin capping. Trans-unsaturated and saturated free fatty acids had no effect. These results were interpreted as being due to perturbation of specific membrane lipid domains. Corps, A. N., Pozzan, T., Hesketh, T. R., and Metcalfe, J. C. (1980) J. Biol. Chem. 255, 10566-10568) in a recent paper argued, however, that linoleic acid causes depletion of ATP, as measured by the luciferin/luciferase assay, and respiratory uncoupling. We now present data showing that under the same conditions as the capping inhibition, there is neither ATP depletion, as found using a hexokinase/glucose-6-phosphate dehydrogenase assay, nor respiratory uncoupling. Linoleic acid produces artifacts in ATP measurements made using the luciferin/luciferase assay which leads to an erroneous conclusion regarding the ATP dependence of the capping inhibition.
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PMID:Inhibition of cap formation on lymphocytes by free fatty acids is not mediated by a depletion of ATP. 706 13

The atractyloside-insensitive accumulation of adenine nucleotides by rat liver mitochondria (as opposed to the exchange-diffusion catalysed by the adenine nucleotide translocase) has been measured by using the luciferin/luciferase assay as well as by measuring [14C]ATP uptake. In foetal rat liver mitochondria ATP is accumulated more rapidly than ADP, whereas AMP is not taken up. The uptake of ATP occurs against a concentration gradient, and the rate of ATP uptake is greater in foetal than in adult rat liver mitochondria. The accumulated [14C]ATP is shown to be present within the mitochondrial matrix space and is freely available to the adenine nucleotide translocase for exchange with ATP present in the external medium. The uptake is specific for ATP and ADP and is not inhibited by adenosine 5'-[beta gamma-imido] triphosphate, GTP, CTP, cyclic AMP or Pi, whereas dATP and AMP do inhibit ATP accumulation. The ATP accumulation is also inhibited by carbonyl cyanide m-chlorophenylhydrazone, KCN and mersalyl but is insensitive to atractyloside. The ATP uptake is concentration-dependent and exhibits Michaelis-Menten kinetics. The divalent cations Mg2+ and Ca2+ greatly enhance ATP accumulation, and the presence of hexokinase inhibits the uptake of ATP by foetal rat liver mitochondria. These latter effects provide an explanation for the low adenine nucleotide content of foetal rat liver mitochondria and the rapid increase that occurs in the mitochondrial adenine nucleotide concentration in vivo immediately after birth.
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PMID:The transport and accumulation of adenine nucleotides during mitochondrial biogenesis. 730 14

A genomic clone containing sequence identical to the 5' region of the cDNA for rat Type I hexokinase was isolated from a lambda Charon 4A library. A 5.4-kb EcoRI fragment from this clone, containing the matching sequence, was sequenced in its entirety. Rapid amplification of 5' cDNA ends (5' RACE), reverse transcription polymerase chain reaction, and ribonuclease protection experiments were consistent with the existence of multiple transcriptional start sites clustered in three regions approximately 460, 300, and 100 nucleotides upstream from the translational start codon. Together with results of previous work, the 5' untranslated sequence defined in the present study accounts for the 4.3-kb mRNA for Type I hexokinase seen on Northern blots. Fragments from the 5' flanking region were cloned into a reporter vector containing the luciferase coding region. Based on transfection experiments with both PC12 and H9c2 cells, promoter activity was associated with a region lying between nucleotide positions -742 and -516 (with A of the ATG codon at the translational start site defined as +1). The promoter region lacks a TATA sequence and, together with the transcriptional start sites, is located within a GC rich segment (a "CpG island") approximately 1 kb in length. These characteristics have previously been associated with the promoter and transcriptional start sites of genes for "housekeeping enzymes."
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PMID:Isolation of the promoter for Type I hexokinase from rat. 891 47

Multiple transcriptional start sites have been identified for the gene encoding the rat Type I isozyme of hexokinase (White, J.A., Liu, W., and Wilson, J. E., Arch. Biochem. Biophys. 335, 161-172, 1996); these are clustered at positions approximately -460, -300, and -100 relative to the translational start codon (ATG, with A being +1). PC12 cells and H9c2 cells were transfected with luciferase reporter constructs containing genomic sequence between positions -3366 and -171. Marked (85%) decrease in promoter activity was associated with deletion of sequence between -742 and -516. In DNase I footprinting experiments, two regions, called P1 (-552 to -529) and P2 (-480 to -458) boxes, were protected by proteins present in nuclear extracts from PC12 cells. Mutation or deletion of the P2 box had no effect on promoter activity; protection in this region, which includes the most upstream cluster of transcriptional start sites, is attributed to binding of RNA polymerase II or associated factors. In contrast, mutations or deletions in the P1 box had markedly detrimental effects on promoter activity and on binding of proteins in PC12 cell nuclear extracts. Maintenance of a consensus Sp1 binding site centrally located in the P1 box was critical for both promoter activity and binding. A second Sp1 site (-570), just upstream from the P1 box, was also shown to be functionally important but no protection of this region was detected in footprinting experiments, presumably reflecting lower affinity at this site under the conditions used. Supershift experiments demonstrated the involvement of Sp1, Sp3, and Sp4 in formation of complexes with the P1 box region and implicate these transcription factors in regulating promoter activity associated with this region. Another series of reporter constructs, including sequence between -171 and -1, permitted detection of an additional promoter activity downstream from -364. While not yet extensively characterized, it is already evident that the cis elements influencing the downstream promoter activity are distinct from the Sp factors determined to be important in expression from the upstream promoter region.
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PMID:Two Sp sites are important cis elements regulating the upstream promoter region of the gene for rat type I hexokinase. 932 94

The concentration of ATP generated by yeast mitochondria and consumed by yeast hexokinase was monitored using native firefly luciferase in solution, or recombinant luciferase localized at the surface of mitochondria. In the absence of hexokinase, both probes perform similarly in detecting exogenous or mitochondrially-generated ATP. The steady-state concentrations of ATP can be reduced in a dose-dependent manner by hexokinase. With hexokinase added in large excess, the localized probe reports substantial ATP concentrations while none is detectable by soluble luciferase. Thus, ATP accumulates near the membrane where it appears, relatively to solution, and vice versa for ADP. The extent of nucleotide gradients is shown to be correlated with the specific activity of oxidative phosphorylation and with the viscosity of the medium, but independent of the concentration of the organelles. A simple model involving diffusional restrictions is presented to describe this behavior. The metabolic and evolutionary implications of cellular catalysis limitation by physical processes are discussed.
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PMID:Localized firefly luciferase probes ATP at the surface of mitochondria. 955 56

The cystic fibrosis (CF) transmembrane regulator (CFTR) is a cyclic AMP-dependent Cl- channel that is defective in CF cells. It has been hypothesized that CFTR exhibits an ATP release function that controls the airway surface ATP concentrations. In airway epithelial cells, CFTR-independent Ca2+-activated Cl- conductance is regulated by the P2Y2 receptor. Thus, ATP may function as an autocrine signaling factor promoting Cl- secretion in normal but not CF epithelia if ATP release is defective. We have tested for CFTR-dependent ATP release using four independent detection systems. First, a luciferase assay detected no differences in ATP concentrations in the medium from control versus cyclic AMP-stimulated primary normal human nasal epithelial (HNE) cells. A marked accumulation of extracellular ATP resulted from mechanical stimulation effected by a medium displacement. Second, high pressure liquid chromatography analysis of 3H-labeled species released from [3H]adenine-loaded HNE cells revealed no differences between basal and cyclic AMP-stimulated cells. Mechanical stimulation of HNE cells again resulted in enhanced accumulation of extracellular [3H]ATP and [3H]ADP. Third, when measuring ATP concentrations via nucleoside diphosphokinase-catalyzed phosphorylation of [alpha-33P]dADP, equivalent formation of [33P]dATP was observed in the media of control and cyclic AMP-stimulated HNE cells and nasal epithelial cells from wild-type and CF mice. Mechanically stimulated [33P]dATP formation was similar in both cell types. Fourth, 1321N1 cells stably expressing the human P2Y2 receptor were used as a reporter system for detection of ATP via P2Y2 receptor-promoted formation of [3H]inositol phosphates. Basal [3H]inositol phosphate accumulation was of the same magnitude in control and CFTR-transduced cells, and no change was observed following addition of forskolin and isoproterenol. In both cell types, mechanical stimulation resulted in hexokinase-attenuable [3H]inositol phosphate formation. In summary, our data suggest that ATP release may be triggered by mechanical stimulation of cell surfaces. No evidence was found supporting a role for CFTR in the release of ATP.
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PMID:Cystic fibrosis transmembrane regulator-independent release of ATP. Its implications for the regulation of P2Y2 receptors in airway epithelia. 959 57

A 1532-base pair 5'-flanking region of the gene encoding rat type III hexokinase has been cloned and sequenced. The total sequence includes positions -1548 to -17 (A of the translational start ATG as position +1). Using luciferase reporter constructs transfected into PC12 (rat pheochromocytoma) and L2 (rat lung) cells, basal promoter activity has been associated with sequence between -182 and -89. This includes a single transcriptional start site, an adenine at position -134 identified by primer extension. Together with previously cloned cDNA sequence, this accounts for an mRNA of approximately 3.9 kilobases, found by Northern blotting of RNA from rat lung and kidney. Sequence upstream of the transcriptional start site was devoid of canonical TATA and CAAT elements. An octamer 1 (Oct-1) binding site, located between positions -166 and -159 was shown by deletion analysis and site-directed mutation to be critical for promoter activity. Nuclear extracts from PC12 cells contained a protein (or proteins) specifically binding the octamer sequence, and supershift experiments with anti-Oct-1 indicated involvement of this ubiquitously expressed transcription factor in the complex. Sequence including the Oct-1 site and immediately adjacent regions was protected from DNase I digestion in footprinting experiments with nuclear extracts from PC12 cells. Reverse transcription polymerase chain reaction indicated that levels of type III hexokinase mRNA in rat tissues increased in the order brain < liver < lung approximately kidney; immunoblotting indicated that type III hexokinase protein in these tissues increased in a similar manner.
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PMID:Characterization of the rat type III hexokinase gene promoter. A functional octamer 1 motif is critical for basal promoter activity. 1053 80


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