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 new adenine nucleotide analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), has been synthesized. The effectiveness of PLP-AMP as an affinity probe has been tested using a number of nucleotide-binding enzymes. In comparison to reaction with pyridoxal 5'-phosphate, PLP-AMP binds more tightly and exhibits greater specificity of labeling for most enzymes tested. PLP-AMP is a very potent inhibitor of yeast alcohol dehydrogenase and rabbit muscle pyruvate kinase, with complete inhibition obtained upon incorporation of 1 mol of reagent/mol of catalytic subunit. The reagent is also a potent inhibitor of yeast hexokinase and phosphoglycerate kinase. When modified in the absence of substrates, these enzymes require 2 mol of reagent/mol of active site for complete inhibition. However, when modified in the presence of sugar substrates, this stoichiometry decreases to 1.1 for the hexokinase-glucose complex and 1.4 for the phosphoglycerate kinase . 3-phosphoglycerate complex. The most potent inhibition by PLP-AMP was observed with rabbit muscle adenylate kinase. Half-maximal inhibition was obtained at a concentration of approximately 1 microM. In contrast to these examples, PLP-AMP, as well as pyridoxal 5'-phosphate, fails to act as a potent or specific inhibitor of beef heart mitochondrial F1-ATP-ase. The high specificity of labeling and the ability of nucleotide substrates to decrease the rate of inactivation of the kinases and dehydrogenase are consistent with the modification of active site residues. The complete reversibility of both modification and inactivation in the absence of reduction by NaBH4 and the absorption spectra of modified enzymes prior to and following reduction indicate reaction with lysyl residues. We conclude that PLP-AMP holds considerable promise as an affinity label for exploring the structure and mechanism of nucleotide-binding enzymes.
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PMID:Affinity labeling of nucleotide-binding sites on kinases and dehydrogenases by pyridoxal 5'-diphospho-5'-adenosine. 293 39

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

Male Jcl: Wistar rats were exposed to 2, 4, and 10 ppm NO2 for 14, 10, and 7 d, respectively, to examine the effect of NO2 on the lung glycolytic pathway, a major energy-generating system in the lung. A highly significant increase in the activities of hexokinase, phosphofructokinase, 3-phosphoglycerate kinase, pyruvate kinase (PK), and lactate dehydrogenase was observed after 5 d exposure to 10 ppm NO2, and a significantly higher value was maintained until d 7. Similarly, the activities of all enzymes examined increased significantly by exposure to 4 ppm NO2, reaching the maximum between 4 and 7 d of exposure, and then approached to near the control levels. The most remarkable increase was found in the PK activity, which reached 1.82- (p less than 0.001) and 1.53-fold (p less than 0.001) that of the control at d 5 (10 ppm) and d 7 (4 ppm) of exposure, respectively. Upon exposure to 2 ppm NO2, the PK activity of exposed animals was also increased to 1.23-fold (p less than 0.05) that of the control at d 7, and a higher activity was maintained until d 14. The glucose-6-phosphate dehydrogenase activity of exposed animals increased significantly at d 3, 4, and 14 of exposures to 10, 4, and 2 ppm NO2, respectively, and a significantly higher value was maintained in the following period of exposure. These results show that short-term exposure of rats to 2-10 ppm NO2 induces the pulmonary systems concerning glycolysis and NADPH-generation. The generation of energy and NADPH in the lung may be enhanced by NO2 inhalation.
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PMID:An increase in the activities of glycolytic enzymes in rat lungs produced by nitrogen dioxide. 315 9

A latex phagocytosis technique was used to prepare relatively pure plasma membranes with inside-out orientation. This method was adapted through a number of modifications in order to evaluate the association of glycolytic enzymes with the cytoplasmic side of the plasma membrane of C6 glial cells. As phosphorylation is strictly coupled with transport in these cells, glycolytic enzymes, especially hexokinase, could metabolize glucose in close vicinity to its transporter. Of the enzymes tested, hexokinase is present in considerable quantities on these membranes (nearly 40% of homogenate specific activity), followed by D-glyceraldehyde-3-phosphate dehydrogenase (10%), pyruvate kinase (8%), and 3-phosphoglycerate kinase (1%). Except for hexokinase, the enzyme pattern presented here is different from that published for other membrane preparations.
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PMID:Association of glycolytic enzymes with the cytoplasmic side of the plasma membrane of glioma cells. 335 18

Enthalpies of binding of MgADP, MgATP, and 3-phosphoglycerate to yeast phosphoglycerate kinase have been determined by flow calorimetry at 9.95-32.00 degrees C. Combination of these data with published dissociation constants [Scopes, R.K. (1978) Eur. J. Biochem. 91, 119-129] yielded the following thermodynamic parameters for the binding of 3-phosphoglycerate at 25 degrees C: delta Go = -6.76 +/- 0.11 kcal mol-1, delta H = 3.74 +/- 0.08 kcal mol-1, delta So = 35.2 +/- 0.6 cal K-1 mol-1, and delta Cp = 0.12 +/- 0.32 kcal K-1 mol-1. The thermal unfolding of phosphoglycerate kinase in the absence and presence of the ligands listed above was studied by differential scanning calorimetry. The temperature of half-completion, t 1/2, of the denaturation and the denaturational enthalpy are increased by the binding of the ligands, the increase in t 1/2 being a manifestation of Le Chatelier's principle and that in enthalpy reflecting the enthalpy of dissociation of the ligand. Only one denaturational peak was observed under all conditions, and in contrast with the case of yeast hexokinase [Takahashi, K., Casey, J.L., & Sturtevant, J.M. (1981) Biochemistry 20, 4693-4697], no definitive evidence for the unfolding of more than one domain was obtained.
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PMID:Thermodynamic study of yeast phosphoglycerate kinase. 354 15

In the process of defining the recruitment of fuel and pathway selection in rainbow trout fast-twitch white skeletal muscle, it was clear that the near-maximal myosin adenosinetriphosphatase activity during a 10-s sprint was supported solely by phosphocreatine hydrolysis. A conservative estimate of the ATP turnover was 188 mumol X g wet wt-1 X min-1. It was not until the rate and force of contraction decreased that the relative contribution of anaerobic glycogenolysis became increasingly important. Over a 10-min period of burst swimming at approximately 120% of maximum aerobic steady-state swimming velocity of trout determined in a Brett-type swim tunnel, fatigue was associated with the near-depletion of glycogen in white muscle. The ATP turnover supported by anaerobic glycogenolysis was 78 mumol X g wet wt-1 X min-1. The glycolytic pathway appeared functional at this time with control sites being identified at hexokinase and phosphofructokinase (PFK-1). PFK-1 did not appear to be inhibited by low muscle pH (pH 6.66). In another exercise protocol lasting 30 min, complete exhaustion was related to glycogen depletion. The sum of all glycolytic intermediates from glucose 6-phosphate to pyruvate at exhaustion decreased by a dramatic 80% compared with the 25% decrease for the 10-min fatigue swimming protocol. This large depletion of glycolytic intermediates was accompanied by an 80% fall in ATP, a 70-80% reduction in the ATP/ADP and phosphorylation potential, and a 2.5-fold increase in the NAD/NADH. Associated with these changes was a marked displacement of the phosphoglycerate kinase (PGK), and the combined glyceraldehyde-3-phosphate dehydrogenase-PGK reactions from thermodynamic equilibrium. As a general conclusion, fatigue and exhaustion should be viewed as a multicomponent biochemical process in response to low glycogen and not leveled at one particular step of the glycolytic pathway.
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PMID:Regulation of anaerobic ATP-generating pathways in trout fast-twitch skeletal muscle. 360 83

The glucose consumption rate versus ATP content in human red cells (regulatory patterns of glycolysis) and ATP concentration versus glucose uptake rate in red cell suspension (regulatory patterns of total ATPases), when the rate of glucose uptake is constant and lower than the rate of glucose consumption at physiological conditions, were measured at different pH values. The shape of both types of kinetic curves was found to be dependent on the pH of the incubation medium but the same for the red cells taken from different donors. It is supposed that at alkaline pH, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase reactions become the rate-limiting steps of glycolysis instead of hexokinase and phosphofructokinase under physiological conditions.
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PMID:[Effect of pH on the regulatory characteristics of energy metabolism in human erythrocytes]. 376 40

The glycosomes of in vitro grown procyclic trypomastigote forms of Trypanosoma brucei were purified by three different procedures and the results compared by electron microscopy, enzyme assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Centrifugation on a self-forming Percoll gradient followed by a sucrose gradient centrifugation resulted in the least enriched glycosomal preparation. Centrifugation on a pre-formed Nycodenz gradient gave an improved preparation but the most homogeneous preparation of intact glycosomes was obtained after centrifugation on two successive sucrose gradients. Glycosomes purified by both the Nycodenz and double sucrose gradient procedures appeared larger than in situ glycosomes presumably due to an osmotic effect resulting from disruption of the granular matrix of the organelles. Nevertheless, there appears to be no loss of cisternal contents due to the swelling of the organelles. The glycosomes of the bloodstream form trypomastigotes purified by the same procedures show, however, no sign of swelling. A comparison of glycosomes purified from procyclic trypomastigotes and bloodstream form trypomastigotes prepared by the same double sucrose procedure demonstrated that in the glycosome of procyclic trypomastigotes: activities of hexokinase, phosphoglucose isomerase, phosphofructose kinase, aldolase and phosphoglycerate kinase and diminished by 80-100%; activities of glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase and glycerol-3-phosphate dehydrogenase remain unchanged or are only slightly reduced; there is an appearance of four major new proteins, among which could be phosphoenol pyruvate carboxykinase and malate dehydrogenase. These observations are in basic agreement with those by Hart et al. (Mol. Biochem. Parasitol. 12, 25-35, 1984).
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PMID:An improved purification of glycosomes from the procyclic trypomastigotes of Trypanosoma brucei. 380 43

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

Glycosomes, the microbody-like organelles containing mainly glycolytic enzymes, were purified from the long slender bloodstream form of Trypanosoma brucei EATRO 110 monomorphic strain by an improved method in which the protozoa were frozen and thawed in 15% glycerol to free, from the plasma membrane, much of the variant surface glycoprotein which used to constitute the major contaminant of our purified glycosomes. The purified glycosomes have 11 major proteins, 6 of which, tentatively identified as phosphofructose kinase, hexokinase, 3-phosphoglycerate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alpha-glycerophosphate dehydrogenase, constitute 87% of the total glycosomal protein. The bifunctional cross-linking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate can penetrate the glycosomal membrane and cause extensive cross-linking of all the major glycosomal proteins. The cross-linked complex, insoluble in 0.1% Triton X-100 plus 0.15 M NaCl, contains all the glycosomal enzyme activities with only partial inactivations. All the enzymes are probably cross-linked into one large complex since they all sediment rapidly to the bottom of a 5-20% (v/v) sucrose density gradient. This successful cross-linking with reagents of span lengths of 11-12 A suggests close proximities among the glycosomal enzymes which may explain the extraordinarily high rate of glycolysis in T. brucei. Whether such a close association represents specific spatial arrangement required for genuine substrate channeling among the enzymes will be verified by future kinetic studies of the cross-linked enzyme complex.
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PMID:Cross-linking of the enzymes in the glycosome of Trypanosoma brucei. 399 56


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