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)

Small-bore ("Autozyme") tubes with immobilized enzymes at the inner wall have been developed and studied for application in the Technicon "SMAC" high-speed continuous-flow biochemical analyzer. Tubes coated with glucose oxidase (D-glucose:oxygen oxidoreductase, EC 1.1.3.4) have been prepared for the assay of glucose, with colorimetric assay of the hydrogen peroxide produced; tubes coated with glycerol kinase (ATP:glycerol phosphotransferase, EC 2.7.1.30) for the enzymatic assay of triglycerides; tubes coated with hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NAD+ oxidoreductase EC 1.1.1.49) for the measurement of ATP, an intermediate product in assays for creatine kinase. With use of 10-15 cm lengths of Autozyme tube and SMAC hydraulics (150 samples per hour), assay sensitivity and carryover were similar to values for the corresponding free-enzyme methods. These immobilized enzymes were sufficiently stable for one to eight weeks of continuous use before replacemnt. We conclude that suitable bound-enzyme tubes can replace either single or multiple free-enzyme reagents in many continuous-flow assays at high sampling rates.
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PMID:Continuous-flow analysis for glucose, triglycerides, and ATP with immobilized enzymes in tubular form. 1 65

The 2-[18O]phosphorothioate of D-glycerate, chiral at phosphorus, was prepared. The chiral phosphoryl group was transferred enzymically to ADP [by using enolase and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase; EC 2.7.1.40)] resulting in the synthesis of adenosine 5'-O-([gamma-18O],gamma-thio)triphosphate. This labeled ATP was used as a thiophosphoryl group donor in the reactions catalyzed by glycerol kinase (ATP:glycerol 3-phosphotransferase; EC 2.7.1.30) and by hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1). The product from the latter (glucose 6-phosphorothioate) was converted enzymically into glycerol phosphorothioate. Determination of the relative configurations and diastereoisomeric purities of the samples of glycerol phosphorothioate demonstrates that all three phosphokinases (pyruvate kinase, glycerol kinase, and hexokinase) transfer the thiophosphoryl group with complete stereospecificity, and further shows that these reactions follow an identical stereochemical course.
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PMID:Adenosine 5'-O-([gamma-18O]gamma-thio)triphosphate chiral at the gamma-phosphorus: stereochemical consequences of reactions catalyzed by pyruvate kinase, glycerol kinase, and hexokinase. 20 59

We report the synthesis of adenosine [gamma-(S)-16O,17O,18O]triphosphate, an isotopically labeled species of ATP that is chiral at the gamma-phosphoryl group, the configuration of which has been confirmed by independent stereochemical analysis. This molecule has been used as a substrate in the reactions catalyzed by glycerol kinase and by acetate kinase. The resulting samples of isotopically labeled sn-glycerol 3-phosphate and of acetyl phosphate have been used as substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl groups to (S)-propane-1,2-diol, whence the configuration at phosphorus has been determined [Abbott, S. J., Jones, S. R., Weinman, S. A., & Knowles, J. R. (1978) J. Am. Chem. Soc. 100, 2558]. It is shown that glycerol kinase and acetate kinase (and, by virtue of an earlier correlation, pyruvate kinase and hexokinase) proceed by pathways that result in inversion of the configuration at phosphorus. The sterochemical approach provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.
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PMID:Stereochemical course of phosphokinases. The use of adenosine [gamma-(S)-16O,17O,18O]triphosphate and the mechanistic consequences for the reactions catalyzed by glycerol kinase, hexokinase, pyruvate kinase, and acetate kinase. 22 19

Kinetic enzymatic methods for analysis of substrates can be made optimum for a sensitive photometric analyzer by adjusting the activity of the triggering (catalyzing) enzyme so that the reaction rate is maximum at the time of measurement. tat this optimum activity, the exponential time constant for exhaustion of substrate equals the time between triggering and rate measurement. The scale factor (defined as measured activity divided by sample concentration in the reaction mixture) is the same for all tests. Sensitivity to substrate concentration is predictable from instrumental absorbance uncertainty and molar absorptivity of the absorbing species. These predictions from Michaelis theory were verified experimentally for pyruvate and lactate triggered with lactate dehydrogenase, for glucose triggered with hexokinase, and for triglycerides triggered with glycerol kinase, the reaction rate being measured 30 s after triggering. Sensitivities of 1.5 times 10(-7) mol/liter were achieved. Serum diluted 1000-fold and analyzed for glucose gave a repeatability of 25 mg/liter with linearity to 4.0 g/liter. Samples diluted 300-fold and analyzed for triglycerides gave 30 mg/liter repeatability, with linearity to concentrations exceeding 3.0 g/liter.
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PMID:Making enzymatic methods optimum for measuring compounds with a kinetic analyzer. 114 29

Phytomonas sp. isolated from Euphorbia characias was adapted to SDM-79 medium. Cells isolated in the early stationary phase of growth were analyzed for their capacity to utilize plant carbohydrates for their energy requirements. The cellulose-degrading enzymes amylase, amylomaltase, invertase, carboxymethylcellulase, and the pectin-degrading enzymes polygalacturonase and oligo-D-galactosiduronate lyase were present in Phytomonas sp. and were all, except for amylomaltase, excreted into the external medium. Glucose, fructose and mannose served as the major energy substrates. Catabolism of carbohydrates occurred mainly via aerobic glycolysis according to the Embden-Meyerhof pathway, of which all the enzymes were detected. Likewise, the end-products of glycolysis, acetate and pyruvate, glycerol, succinate and ethanol were detected in the culture medium, as were the enzymes responsible for their production. Mitochondria were incapable of oxidizing succinate, 2-oxoglutarate, pyruvate, malate and proline, but had a high capacity to oxidize glycerol 3-phosphate. This oxidation was completely inhibited by salicylhydroxamic acid. No cytochromes could be detected either in intact mitochondria or in sub-mitochondrial particles. Mitochondrial respiration was not inhibited by antimycin, azide or cyanide. The glycolytic enzymes, from hexokinase to phosphoglycerate kinase, and the enzymes glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase and adenylate kinase, were all associated with glycosomes that had a buoyant density of about 1.24 g cm-1 in sucrose. Cytochemical staining revealed the presence of catalase in these organelles. The cytosolic enzyme pyruvate kinase was activated by fructose 2,6-bisphosphate, typical of all other pyruvate kinases from Kinetoplastida. The energy metabolism of the plant parasite Phytomonas sp. isolated from E. characias resembled that of the bloodstream form of the mammalian parasite Trypanosoma brucei.
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PMID:Characterization of carbohydrate metabolism and demonstration of glycosomes in a Phytomonas sp. isolated from Euphorbia characias. 143 59

Intracellular phosphorylation is an important step in active uptake and utilization of carbohydrates. For example glucose and glycerol enter the liver cell along the extra intracellular gradient by facilitated diffusion through specific carriers and are concentrated inside the cell by phosphorylation via hexokinase or glycerol kinase. Depending on the function of the respective tissue the uptake of carbohydrates serves different metabolic purposes. In brain and kidney medulla cells which depend on carbohydrates, glucose and glycerol are taken up according to the energy demand. However, in tissues such as muscle which synthesize glycogen or like liver which additionally produce fat from glucose, the uptake of carbohydrates has to be regulated according to the availability of glucose and glycerol. How the reversible coupling of the kinases to the outer membrane pore and the mitochondrial ATP serves to fulfil these specific requirements will be explained as well as how this regulates the carbohydrate uptake in brain according to the activity of the oxidative phosphorylation and how this allows glucose uptake in liver and muscle to persist in the presence of high glucose 6-phosphate without activating the rate of glycolysis.
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PMID:Interaction of mitochondrial porin with cytosolic proteins. 168 54

Porin is the pore-forming protein involved in the movement of adenine nucleotides across the outer mitochondrial membrane (OMM). Hexokinase and glycerol kinase interact with porin on the outer surface of the OMM in a manner which provides these enzymes with preferred access to the ATP generated in the mitochondrion. We review recent evidence which permits refinement of our knowledge of these proteins and their interactions at the OMM. The involvement of this system in metabolic microcompartmentation is discussed, as well as possible pathological consequences of its disruption in malignancy and genetic deficiencies of hexokinase, glycerol kinase, and porin.
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PMID:Porin interaction with hexokinase and glycerol kinase: metabolic microcompartmentation at the outer mitochondrial membrane. 171 Sep 14

Interactions between intramitochondrial ATP-generating, ADP-requiring processes and ATP-requiring, ADP-generating phosphorylation of glucose by mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) have been investigated using well-coupled mitochondria isolated from rat brain. ADP generated by mitochondrially bound hexokinase was more effective at stimulating respiration than was ADP generated by hexokinase dissociated from the mitochondria, and pyruvate kinase was less effective as a scavenger of ADP generated by the mitochondrially bound hexokinase than was the case with ADP generated by the dissociated enzyme. These results indicate that ADP generated by the mitochondrially bound enzyme is at least partially sequestered and directed toward the mitochondrial oxidative phosphorylation apparatus. Under the conditions of these experiments, the maximum rate of ATP production by oxidative phosphorylation was approximately 10-fold greater than the maximum rate of ATP generation by the adenylate kinase reaction. Moreover, during periods of active oxidative phosphorylation, adenylate kinase made no detectable contribution to ATP production. Thus, adenylate kinase does not represent a major source of ATP for hexokinase bound to actively phosphorylating brain mitochondria. With adenylate kinase as the sole source of ATP, a steady state was attained in which ATP formation was balanced by utilization in the hexokinase reaction. In contrast, when oxidative phosphorylation was the source of ATP, a steady state rate of Glc phosphorylation was attained, but it was equivalent to only about 40-50% of the rate of ATP production and thus there was a continued net increase in ATP concentration in the system. Rates of Glc phosphorylation with ATP generated by oxidative phosphorylation exceeded those seen with equivalent levels of exogenously added ATP. Moreover, at total ATP concentrations greater than approximately 0.2 mM, hexokinase bound to actively phosphorylating mitochondria was unresponsive to continued slow increases in ATP levels; acute increase in ATP (by addition of exogenous nucleotide) did, however, result in increased hexokinase activity. The relative insensitivity of mitochondrially bound hexokinase to extramitochondrial ATP suggested dependence on an intramitochondrial pool (or pools) of ATP during active oxidative phosphorylation. Two intramitochondrial compartments of ATP were identified based on their selective release by inhibitors of electron transport or oxidative phosphorylation. These compartments were distinguished by their sensitivity to inhibitors and the kinetics with which they were filled with ATP generated by oxidative phosphorylation. Exogenous glycerol kinase competed effectively with mitochondrially bound hexokinase for extramitochondrial ATP, with relatively low levels of glycerol kinase completely inhibiting phosphorylation of Glc.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Hexokinase of rat brain mitochondria: relative importance of adenylate kinase and oxidative phosphorylation as sources of substrate ATP, and interaction with intramitochondrial compartments of ATP and ADP. 189 45

Glucose uptake and metabolism in the bloodstream form of the glycosome-containing protozoan parasite Trypanosoma brucei was measured using 14C-labeled glucose in combination with the silicone oil centrifugation technique in short term (5-60 s) incubations. Glucose rather than glucose analogues was used to study the interrelation between the uptake process and the subsequent metabolic steps. Glucose uptake over the plasma membrane occurred by facilitated diffusion, which limited the overall glycolytic rate at external glucose concentrations (glcout) below 5 mM. At higher glcout another step, either transport over the glycosomal membrane or phosphorylation by hexokinase became rate-limiting. Mathematical modeling assuming that glucose uptake occurs by facilitated diffusion followed by an enzymatic step accurately predicts the experimental data. As predicted by the model, the internal concentration of non-metabolized glucose remains low till glcout = 5 mM and increases at higher external concentrations. In contrast to glucose, glycerol entered the cell by simple diffusion. Externally supplied glycerol did not affect glucose metabolism but externally added glucose interfered with glycerol metabolism in a way that suggests that the rate-limiting step is at the level of glycerol kinase. Our observations suggest that the bloodstream form of T. brucei adapts its glucose transport in a way that gives maximum yield at minimum expense.
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PMID:Glucose uptake by Trypanosoma brucei. Rate-limiting steps in glycolysis and regulation of the glycolytic flux. 198 67

The porins are a class of voltage-dependent, anion-selective, channel-forming proteins located in the outer mitochondrial membrane (OMM). The porins are responsible for passage of adenine nucleotides across the OMM, as well as for specific binding of hexokinase and glycerol kinase. This porin-kinase complex has direct access to ATP generated by mitochondrial oxidative phosphorylation and may be important in the regulation of glycolysis. Porin had not been described previously in humans but, due to its importance in bioenergetics, would be expected to be present, especially in organs requiring a large and constant supply of energy. We therefore postulated that porin would occur in human myocardium where it would be important in cardiac function. Polyclonal antibodies to bovine myocardial and rat liver porins were utilized in transblotting experiments after polyacrylamide gel electrophoresis of human heart preparations from atria, ventricles, papillary muscles, and interventricular septum. These immunoblots demonstrated selective staining of a 34-kDa band. This was identical to the results obtained with purified porin and the antibodies. Also notable was the finding that the vast majority of this staining was found in the homogenate pellet after high speed centrifugation (20,000g), as would be expected for a mitochondrial protein. The demonstration of human cardiac porin by immunoblotting with rat liver and bovine myocardial porin antibodies is the first demonstration of cross-species identification of the porins. The success of this approach undoubtedly occurred because of strong homology between porins from a variety of species.
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PMID:Demonstration and characterization of human cardiac porin: a voltage-dependent channel involved in adenine nucleotide movement across the outer mitochondrial membrane. 247 50


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