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)

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

A new method that permits rapid, sensitive, and specific enzymatic assay of proteins in polyacrylamide gels is described. The enzyme product blot described in this report involves percolation of the reaction mixture through a gel containing native enzyme which converts the labeled substrate to a labeled product with differing chemical properties. A permeable membrane with specific ligand-binding properties overlies the gel and binds the enzyme product, but not the substrate, as reaction mixture is blotted vertically. This membrane is washed free of substrate and the location of the product is identified by autoradiography. The autoradiogram is compared with the stained gel in order to recover the enzyme for amino acid sequence analysis. The enzyme product blot is demonstrated using glycerol kinase and hexokinase.
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PMID:Enzyme product blot for nondestructive assay of protein catalytic function in polyacrylamide gels. 272 76

We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus (1971, J. Biol. Chem. 246, 3885-3894) for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays.
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PMID:Radiometric assays for glycerol, glucose, and glycogen. 281 33

We have developed a method for the simultaneous purification of hexokinase, glucosephosphate isomerase, phosphofructokinase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase, D-glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, glycerol-3-phosphate dehydrogenase and glycerol kinase from Trypanosoma brucei in yields varying over 8-55%. Crude glycosomes were prepared by differential centrifugation of cell homogenates. Subsequent hydrophobic interaction chromatography on phenyl-Sepharose resulted in six pools containing various mixtures of enzymes. These pools were processed via affinity chromatography (immobilized ATP), hydrophobic interaction chromatography (octyl-Sepharose) and ion-exchange chromatography (CM- and DEAE-cellulose) which resulted in the purification of all nine enzymes. The native enzyme and subunit molecular masses, as determined by gel filtration and gel electrophoresis under denaturing conditions, were compared with those of their homologous counterparts from other organisms. Trypanosomal hexokinase is a hexamer and differs in subunit composition from the mammalian enzymes (monomers) as well as in subunit size (51 kDa versus 96-100 kDa, respectively). Phosphofructokinase only differs in subunit size (51 kDa for T. brucei versus 80-90 kDa for mammals) but had identical subunit composition (tetrameric). The others all have the same subunit composition as their mammalian counterparts. Except for triosephosphate isomerase, all Trypanosoma enzymes have subunits which are 1-5 kDa larger in size. Together these nine enzymes contribute 3.3 +/- 1.6% to the total cellular protein of T. brucei and at least 90% to the total glycosomal protein. A comparison of calculated intraglycosomal concentrations of the enzymes with the glycosomal metabolite concentrations shows that in the case of aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, the concentration of active sites is of the same order of magnitude as that of their reactants. A common feature of the glycosomal glycolytic enzymes (with the exception of glucosephosphate isomerase) is that they are highly basic proteins with pI values between 8.8 and 10.2, values which are 1-4 higher than in the case of their mammalian cytosolic counterparts and 3-6 higher than in the case of the various unicellular organisms. It is suggested that both the larger subunit size and the basic character of the T. brucei glycolytic proteins are involved in the routing of the enzymes from their site of biogenesis (the cytosol) towards their site of action (the glycosome).
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PMID:Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties. 294 90

Mitochondrial-bound glycerol kinase in rat brain was examined with reference to factors involved in the binding and significance of the binding in relation to ATP metabolism inside the mitochondria. The mitochondrial-bound glycerol kinase was solubilized with glycerol 3-phosphate or ADP, and the solubilized enzyme was rebound to mitochondria by addition of divalent cations. The rebinding was decreased by the presence of glycerol 3-phosphate, while was increased by glucose 6-phosphate. Positive correlation was found between the formation of glycerol 3-phosphate by mitochondrial-bound glycerol kinase and ATP content in mitochondria in experiments using various concentrations of succinate and ADP. On the other hand, glycerol 3-phosphate formation was inhibited by addition of inhibitors for mitochondria functions, such as oligomycin, dinitrophenol, cyanide, and atractyloside. Furthermore, formation of dihydroxyacetone phosphate from glycerol was proved, indicating the involvement of glycerol kinase in glycerol phosphate shuttle in combination with glycerol phosphate dehydrogenase. These findings are discussed in comparison with those of mitochondrial-bound hexokinase.
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PMID:Binding and function of mitochondrial glycerol kinase in comparison with those of mitochondrial hexokinase. 298 25

Glycosomes, purified from trypomastigote forms of Trypanosoma brucei, contained all the enzymes necessary to convert glucose to alpha-glycerophosphate and 3-phosphoglycerate. The multienzyme reaction which produces 2 alpha-glycerophosphate, 2 ADP, and 2 NAD+ from 1 glucose, 2 ATP, and 2 NADH was studied spectrophotometrically. Intact glycosomes, suspended with 5.6 mM alpha-glycerophosphate and 2 mM ADP, produced ATP inside the glycosomes for glucose phosphorylation at a rate of 0.7 mumol/min/mg protein, so confirming the feasibility of producing ATP from alpha-glycerophosphate and ADP catalyzed by glycosomal glycerol kinase, and coupling this ATP production to the ATP-requiring stages of glycolysis. No evidence was found for direct channeling of the ATP generated by glycerol kinase and either hexokinase or phosphofructose kinase in glycosomal enzyme complexes cross-linked by dimethyl suberimidate treatment of intact glycosomes prior to solubilization of their membrane. Compartmentation of glycolytic intermediates, enzymes, and ATP inside isolated glycosomes was demonstrated by their inaccessibility to exogenous enzymes. We conclude that the compartmentation of the glycosome and the efficient production of ATP in the glycosome from whole cell concentrations of sn-glycerol 3-phosphate and ADP account for the observed whole cell production of equimolar glycerol from glucose with net ATP synthesis by T. brucei under anaerobic conditions.
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PMID:The role of compartmentation and glycerol kinase in the synthesis of ATP within the glycosome of Trypanosoma brucei. 299 27

A study of the reverse reaction of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been performed using a photometric method based on a mutarotase-glucose oxidase-peroxidase-chromogen system to trap and visualize glucose, plus a glycerol kinase-glycerol system to trap ATP. Glucose 6-phosphate or 2-deoxyglucose 6-phosphate were used as phosphoryl donors at different concentrations of ADP. Variation of glucose 6-phosphate concentrations resulted in a biphasic curve from which apparent Km and Ki values of ca. 0.2 mM were calculated. In contrast, variation of 2-deoxyglucose 6-phosphate concentrations resulted in Michaelian kinetics with an apparent Km of 2 mM. The Km value for MgADP was 16 mM irrespective of the nature and concentration of the hexose 6-phosphate substrate. These results are fully consistent with an allosteric site for glucose 6-phosphate as an explanation for the inhibition of animal hexokinases by glucose 6-P and further indicate that the maximal rate is the parameter affected. From these observations and previous knowledge, the possible occurrence in animal hexokinases of a regulatory site for ATP to account for the competition between glucose 6-phosphate and ATP in the forward reaction is postulated.
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PMID:Allosteric inhibition of brain hexokinase by glucose 6-phosphate in the reverse reaction. 400 67

The enzyme activities involved in fructose metabolism were measured in samples of human liver. On the basis of U/g of wet-weight the following results were found: ketohexokinase, 1.23; aldolase (substrate, fructose-1-phosphate), 2.08; aldolase (substrate, fructose-1,6-diphosphate), 3.46; triokinase, 2.07; aldehyde dehydrogenase (substrate, D-glyceraldehyde), 1.04; D-glycerate kinase, 0.13; alcohol dehydrogenase (nicotinamide adenine dinucleotide [NAD]) substrate, D-glyceraldehyde), 3.1; alcohol dehydrogenase (nicotinamide adenine dinucleotide phosphate [NADP]) (substrate, D-glyceraldehyde), 3.6; and glycerol kinase, 0.62. Sorbitol dehydrogenases (25.0 U/g), hexosediphosphatase (4.06 U/g), hexokinase (0.23 U/g), and glucokinase (0.08 U/g) were also measured. Comparing these results with those of the rat liver it becomes clear that the activities of alcohol dehydrogenases (NAD and NADP) in rat liver are higher than those in human liver, and that the values of ketohexokinase, sorbitol dehydrogenases, and hexosediphosphatase in human liver are lower than those values found in rat liver. Human liver contains only traces of glycerate kinase. The rate of fructose uptake from the blood, as described by other investigators, can be based on the activity of ketohexokinase reported in the present paper. In human liver, ketohexokinase is present in a four-fold activity of glucokinase and hexokinase. This result may explain the well-known fact that fructose is metabolized faster than glucose.
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PMID:Enzymes of fructose metabolism in human liver. 438 49


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