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)

A routine assay for CTP in cell and tissue extracts using crude firefly lantern preparations is described. ATP, GTP, and UTP are removed by incubation of the samples with a mixture of 3-phosphoglycerate kinase, hexokinase, glucose-6-phosphate dehydrogenase, and UDP-glucose pyrophosphorylase in the presence of ADP. The method is sensitive (greater than 30 nM CTP), inexpensive, and reproducible. No chromatographic purification of the biological samples or of the firefly extract is necessary. Corrections must be made for some loss of CTP during the enzymatic incubation and for the background luminescence of ADP. The applicability of the assay is tested with extracts from the cyanobacterium Anacystis nidulans.
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PMID:A firefly luciferase assay for determination of cytidine 5'-triphosphate in biological samples. 392 76

Addition of 0.4-25 microM extracellular ATP results in transient, dose-dependent increases in cytosolic free calcium measured in Ehrlich ascites tumor cells. In cells incubated with 1 mM extracellular Ca2+, ATP induces a triphasic Ca2+ transient: an initial rapid increase (2-3 s), a second, slower phase of increase (60-90 s), and, finally, a gradual return to near resting [Ca2+]i (4-5 min). Several findings demonstrate that the initial, rapid phase of Ca2+ transient results from a mobilization of Ca2+ from a non-mitochondrial intracellular store, while the second, slow phase of increase is produced by enhanced influx of Ca2+ across the plasma membrane. Successive additions of extracellular ATP can elicit repetitive Ca2+ transients if the initially added ATP is removed either through the action of native ecto-ATPase activity or exogenous hexokinase. Other adenine nucleotides, including non-hydrolyzable ATP analogs, neither alter cytosolic [Ca2+] nor antagonize the ATP-induced effects. Conversely, other nucleotide triphosphates (ITP, UTP, and GTP) induce Ca2+ transients which are identical to those produced by ATP. A variety of experimental results indicate that these actions of ATP and other nucleotide triphosphates are not due to a generalized increase in plasma membrane permeability. The results suggest that, in these transformed cells, ATP may act in a manner similar to other Ca2+ mobilizing hormones and growth factors.
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PMID:Intracellular Ca2+ mobilization activated by extracellular ATP in Ehrlich ascites tumor cells. 403 Jul 63

1. The substrate kinetic properties of cerebral hexokinases (mitochondrial and cytoplasmic) were studied at limiting concentrations of both glucose and MgATP(2-). Primary plots of the enzymic activity gave no evidence of a Ping Pong mechanism in three types of mitochondrial preparation tested (intact and osmotically disrupted mitochondria, and the purified mitochondrial enzyme), nor in the purified cytoplasmic preparation. 2. Secondary plots of intercepts from the primary plots (1/v versus 1/s) versus reciprocal of second substrate of the mitochondrial activity gave kinetic constants which differed from those obtained directly from the plots of 1/v versus 1/s or of s/v versus s, although the ratios of the derived constants were consistent. The kinetic constants obtained with the cytoplasmic enzyme from primary and secondary plots were consistent. 3. Deoxyglucose, as alternative substrate, inhibited cytoplasmic hexokinase by competition with glucose, but did not compete when MgATP(2-) was the substrate varied. The K(i) for deoxyglucose when glucose concentrations were varied was 0.25mm. 4. A range of ATP analogues was tested as potential substrates and inhibitors of hexokinase activity. GTP, ITP, CTP, UTP and betagamma-methylene-ATP did not act as substrates, nor did they cause significant inhibition. Deoxy-ATP proved to be almost as effective a substrate as ATP. AMP inhibited but did not act as substrate. 5. N-Acetyl-glucosamine inhibited all preparations competitively when glucose was varied and non-competitively when MgATP(2-) was varied. AMP inhibition was competitive when MgATP(2-) was the substrate varied and non-competitive when glucose was varied. 6. The results are interpreted as providing evidence for a random reaction mechanism in all preparations of brain hexokinase, cytoplasmic and mitochondrial. The kinetic properties and reaction mechanism do not change on extraction and purification of the particulate enzyme. 7. The results are discussed in terms of the participation of hexokinase in regulation of cerebral glycolysis.
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PMID:Cerebral-cortex hexokinase. Elucidation of reaction mechanisms by substrate and dead-end inhibitor kinetic analysis. 512 80

Inhibition of IMP dehydrogenase in AS-30D hepatoma cells in suspension culture resulted in a pronounced and selective reduction of guanine nucleotide pools. Total acid-soluble guanine nucleotides decreased to 40% and the content of GTP and GDP dropped to about 20% of control within 4 h when mycophenolate or ribavirin were used as the inhibitors. Induction of GTP deficiency was associated with a 50% rise in UTP and other uracil nucleotides. Guanosine rapidly reversed both the reduction of guanine nucleotide pools and the elevation of cellular UTP contents. Enzymatic nucleotide analyses in cell and tissue extracts after treatment with ribavirin indicated that ribavirin 5'-triphosphate was an effective substrate for yeast hexokinase, yeast phosphoglycerate kinase, and nucleosidediphosphate kinase from yeast or bovine liver. These results were confirmed in detail by the use of synthetic ribavirin 5'-triphosphate and 5'-diphosphate. The latter nucleotide analog was also a substrate of pyruvate kinase from muscle. Mycophenolate-induced GTP deficiency was associated with an arrest of hepatoma cell growth in suspension culture. Ribavirin, at an equimolar concentration, was much less effective in this respect. None of the two inhibitors had a detectable effect, however, in vivo when guanine or uracil nucleotides were assayed in liver. This indicated that an inhibition of de novo guanylate synthesis in vivo can be compensated by salvage pathway synthesis.
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PMID:Selective guanosine phosphate deficiency in hepatoma cells induced by inhibitors of IMP dehydrogenase. 610 11

Deciliation of Paramecium tetraurelia by a Ca2+ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca2+-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca2+, Sr2+, or Ba2+, but not by Mg2+ or by monovalent cations. The crude enzyme has a specific activity of 2-3 mumol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50 degrees C, and which has a sedimentation coefficient of 8-10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.
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PMID:A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia. 612 13

Mg2+-ATPase activity was identified in the cytosol of human erythrocytes. A partial purification of this activity was achieved by an initial DEAE-Sephadex column chromatography, followed by gel filtration on Sephadex G-100 and then a second DEAE-Sephadex chromatography procedure. The enzyme appeared in the void volume of the Sephadex G-100 column and was retained on an Amicon XM100A ultrafiltration membrane. The molecular weight of the enzyme was estimated to be 113 000 from SD gels. The above purification protocol yielded an enzyme with an optimal pH between 7.6 and 8.2. The enzyme activity increased linearly between 30 and 44 degrees C. It was stable for several months at -20 degrees C. Magnesium was essential for activity, but the rate attainable with Mn2+ was at least as great as that due to Mg2+. No other divalent cation was able to substitute for Mg2+ or Mn2+. Neither low nor high Ca2+ concentrations significantly affected the enzymatic activity. Substrate specificity studies showed that ATP was the preferred substrate followed by CTP (46% of the rate produced by ATP). Hydrolysis of GTP, UTP, ITP and ADP was less than 10% of the rate seen with ATP. No phosphatase, pyrophosphatase, phosphodiesterase, hexokinase, phosphofructokinase or adenylate cyclase activity could be detected in this enzyme preparation. Calmodulin, which stimulates the (Ca2+ + Mg2+)-ATPase of the human erythrocyte membrane, failed to enhance the Mg2+-ATPase activity. Of considerable interest, the activity of this Mg2+-ATPase was enhanced approximately 5-fold by low concentrations of mercuric ion, p-hydroxymercuribenzoate and DTNB, but was much less sensitive to iodoacetamide.
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PMID:Partial purification and characterization of a novel Mg2+-dependent ATPase present in the cytosol from human erythrocytes. 615 Jul 30

Hexokinase (ATP: hexose 6-phosphotransferase, E.C.2.7.1.1) and phosphofructokinase (ATP:fructose-6-phosphate 1-phosphotransferase, E.C.2.7.1.11), two key regulatory enzymes of the glycolytic pathway in vertebrate cells, have been isolated and partially purified from Trypanosoma (Schizotrypanum) cruzi epimastigotes. Both enzymes are associated with particles sedimentable at 105 000 X gav for 1 h and have a high degree of latency; they can be solubilized by sonication. Hexokinase catalyses the phosphorylation of a series of monosaccharides at the following relative rates: D-glucose (100) congruent to D-fructose (97) greater than 2-deoxy-D-glucose (72) congruent to mannose (69) greater than 2-amino-D-glucose (63) greater than 3-O-methyl-D-glucose (21). Very little or no phosphorylating activity was found for D-galactose, N-acetyl-2-amino-D-glucose or 1-alpha-methyl-D-glucose. D-Glucose phosphorylation at fixed ATP concentration follows simple Michaelis-Menten kinetics with Km = 40 microM and Vmax = 440 nmol min-1 mg-1 protein. D-Mannose, 2-deoxy-D-glucose and N-acetyl-2-amino-D-glucose act as competitive inhibitors of glucose phosphorylation, suggesting a single kinase. Mg2+-ATP is the preferred phosphoryl donor, ITP and GTP being much less effective. T. cruzi hexokinase is not inhibited by D-glucose 6-phosphate, or by any of the following compounds (2 mM):D-fructose 6-phosphate, D-fructose 1,6-diphosphate, D-glucose 1,6-diphosphate, phosphoenol pyruvate, L-malate and citrate. Phosphofructokinase displays simple Michaelis-Menten kinetics with no evidence of sigmoidicity with respect to D-fructose 6-phosphate at all ATP concentrations tested, giving a Km of 1.31 mM and Vmax = 400 nmol min-1 mg-1 protein at optimal ATP levels. With respect to ATP, the enzyme exhibits Michaelis-Menten kinetics at low concentration (less than 1 mM) of the substrate (Km = 40 microM at 5 mM MgCl2, pH 7.4). A moderate inhibition is observed at high ATP levels (70% of maximal activity at 2 mM). GTP can substitute for ATP as the phosphoryl donor (Km = 79 microM under the same conditions), but produces only very small inhibitory effects at high concentrations. 5'-AMP activates the enzyme by decreasing its Km with respect to D-fructose 6-phosphate without affecting Vm. Other well-known regulators of the activity of this enzyme in procaryote and vertebrate systems such as citrate, phosphoenol pyruvate, ammonium and phosphate ions have no effect in T. cruzi.
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PMID:Regulation of energy metabolism in Trypanosoma (Schizotrypanum) cruzi epimastigotes. I. Hexokinase and phosphofructokinase. 623 52

We examined the hypothesis that initiation of eukaryotic protein synthesis involves ATP-dependent melting of 5'-cap-proximal secondary structure in mRNA by eukaryotic initiation factors 4A and 4B. In reticulocyte lysate depleted of ribonucleoside triphosphates by pretreatment with hexokinase/glucose, initiation complex formation by native reovirus mRNA showed a strict requirement for ATP. The corresponding mRNA synthesized with ITP in place of GTP to minimize secondary structure also required ATP for binding to 40 S ribosomal subunits in complexes characteristic of initiation. In a partial reaction without ribosomes, purified eukaryotic initiation factors 4A and 4B bound and cross-linked to the capped 5'-end of oxidized mRNA. This interaction was ATP-dependent with inosine-substituted or bromouridine-containing reovirus RNAs as observed previously with native mRNA. The results indicate that if initiation involves ATP-dependent denaturation of mRNA, the effect must occur after initiation factor-mediated attachment of mRNA to the 40 S ribosomal subunit.
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PMID:Binding of inosine-substituted mRNA to reticulocyte ribosomes and eukaryotic initiation factors 4A and 4B requires ATP. 688 21

Regulation of glucose metabolism in glycolysis by round spermatids was studied. Assay of activities of 11 glycolytic enzymes in cell-free spermatid extracts showed that hexokinase, phosphofructokinase, and glyceraldehyde-3-phosphate dehydrogenase had the lowest activities. When the cells were incubated with glucose (10 mM), the intracellular level of ATP fell rapidly and 5'-AMP increased. The ADP level remained unchanged. During incubation with glucose, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, and glyceraldehyde-3-phosphate were accumulated without any change in a mass action ratio of fructose bisphosphate aldolase. Glyceraldehyde-3-phosphate dehydrogenase appeared to play a regulatory role in glycolysis. Glyceraldehyde-3-phosphate dehydrogenase was inhibited by the following compounds (Ki values in parentheses): adenosine (4.34 mM), 5'-AMP (3.50 mM), ADP (2.35 mM), ATP (5.34 mM), and 3',5'-cAMP (0.60 mM). In each case, the inhibition was competitive with NAD (Km = 0.20 mM). The 2'-hydroxy group of the adenine-linked ribose moiety was essential for binding. The compounds adenine, 2'-deoxyadenosine, 2'-AMP, 3'-AMP, CTP, GTP, UTP, and NADP showed little inhibition. These findings suggest that regulation of glycolysis in round spermatids by glyceraldehyde-3-phosphate dehydrogenase is most likely and that glyceraldehyde-3-phosphate dehydrogenase is inhibited by the adenine nucleotides, particularly by 5'-AMP and ADP as inhibitors competitive with NAD.
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PMID:Regulation of glucose metabolism by adenine nucleotides in round spermatids from rat testes. 714 87

We have developed a coupled enzyme system composed of hexokinase, glucose, and nucleoside diphosphate kinase which is able to rapidly convert GTP at the exchangeable nucleotide binding site of tubulin to GDP. Using this method, we have studied the dynamic properties of microtubules and tubulin subunits in the presence of GDP. Conversion of GTP to GDP causes microtubules to change to a new, somewhat lower steady state level; dilution studies show that subunits disassemble from microtubules at steady state in the presence of GDP at a rate comparable to that in the presence of GTP; in reactions of existing microtubules with tubulin-GDP subunits it was found that tubulin-GDP subunits do not participate in net microtubule elongation. From these observations we conclude that in the presence of tubulin-GDP the microtubule steady state has an unusual property; in spite of the fact that the microtubule is continuously undergoing rapid subunit loss, but not subunit addition, a nearly constant steady state level of microtubule mass is maintained. This must mean that tubulin-GDP subunits, although unable to participate in a net addition, can participate in additions each of which compensates for the dissociation of a single subunit from the microtubule. This is equivalent to the existence of an isoenergetic exchange of a tubulin-GDP in solution for a subunit which had been lost from the end of a microtubule.
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PMID:An isoenergetic exchange mechanism which accounts for tubulin-GDP stabilization of microtubules. 729 43


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