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)

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

Incubation of [gamma-32P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma-32P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma-32P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide.
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PMID:Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase. 293 42

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

An H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H2O2 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0.1 microM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hexokinase together with glucose to remove ATP. The Km value for NADPH was 35 microM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H2O2 production. The stoichiometry between the oxidation and the H2O2 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2 production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H2O2 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H2O2. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H2O2. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H2O2, a substrate for peroxidase.
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PMID:Activation by ATP of calcium-dependent NADPH-oxidase generating hydrogen peroxide in thyroid plasma membranes. 312 60

Data have been obtained suggesting that the complex porin-hexokinase of brain mitochondria may be related to the contact sites between the outer and inner membrane. In the attempt to isolate from brain mitochondria the inner and outer membranes and the boundary membrane contacts, a procedure was developed based on swelling and shrinking of the organelles, followed by sonication and reverse discontinuous density gradient centrifugation. Three fractions were obtained by this technique, which were identified by measuring the relative specific activities of marker enzymes, namely succinate-cytochrome c reductase; NADH-cytochrome c reductase (rotenone insensitive); hexokinase and glutathione transferase, for the inner and outer membranes and contact sites, respectively. The fraction which contains the contact sites is characterized by the highest specific activity of hexokinase and glutathione transferase and by the highest calcium binding capacity; physiological concentrations of this cation produces a sharper separation of this fraction. Results indicate that both the porin-hexokinase gating system of the outer membrane and the calcium transporting complex of the inner membrane are present in the fraction which contains the contact sites.
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PMID:Influence of Ca2+ on the isolation from rat brain mitochondria of a fraction enriched of boundary membrane contact sites. 319 26

The effect of hypoxia and post-hypoxic recovery were studied in gastrocnemius muscle of young-adult and mature beagle dogs. Furthermore, the possible interference of pharmacological treatment with nicergoline was evaluated in these conditions. Muscular glycolytic fuels, intermediates and end-products (glycogen, glucose, glucose 6-phosphate, pyruvate, lactate), Kreb's cycle intermediates (citrate, alpha-ketoglutarate, succinate, malate) and related free amino acids (glutamate, alanine), ammonium ion, energy store and mediators (ATP, ADP, AMP and creatine phosphate), and the energy charge potential were evaluated. Furthermore, in the crude extract and/or mitochondrial fraction of another portion of the same gastrocnemius muscle the maximum rate (Vmax) of some muscular enzymes related to the anaerobic glycolytic pathway (hexokinase, lactate dehydrogenase), the Kreb's cycle (citrate synthase, malate dehydrogenase), the aminoacid pool related to the Krebs' cycle (glutamate dehydrogenase and aspartate aminotransferase), the electron transfer chain (cytochrome oxidase) and NAD+/NADH exchanges (total NADH cytochrome c reductase) was evaluated. Some glycolytic metabolites and Krebs' cycle intermediates were modified by acute hypoxia, while free amino acids and energy mediators remained practically unchanged. The pharmacological treatment maintained the glucose and succinate muscular concentrations within the normal range, during hypoxia. The behaviour of muscular metabolites during hypoxia and/or post-hypoxic recovery is an age-related event. In fact, only in young-adult animals did the altered values return to normal in post-hypoxic recovery. In the present experimental conditions, only minor changes were observed as far as muscular enzyme activities are concerned. In any case, some enzyme activities tested showed different Vmax in young-adult dogs in comparison with mature ones.
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PMID:Effect of hypoxia, aging and pharmacological treatment on muscular metabolites and enzyme activities. 322 9

The erythrocyte can phosphorylate a variety of hexoses. Since it can consume mannose and glucose equivalently in the hereditary deficiencies of hexokinase and phosphoglucose isomerase and since erythrocyte defense against oxidants is impaired in a variety of hereditary hemolytic anemias, we tested the hypothesis that mannose may be a significant alternative to glucose as a fuel for this defense system. Unexpectedly, mannose inhibited defense against oxidants as manifested by increased Heinz body formation when both normal and high-reticulocyte erythrocytes were incubated with acetylphenylhydrazine (APH). Using APH as the oxidant, mannose-incubated erythrocytes had decreased reduced glutathione stability and impaired hexose oxidation by the pentose shunt compared to glucose-incubated erythrocytes. After incubation with mannose and APH, normal erythrocytes showed a decrease in ATP content. Approximately 25% of the consumed mannose accumulated in the erythrocytes as mannose 6-phosphate. Erythrocytes incubated with mannose and APH displayed a significant loss of redox potential as manifested by decreased NADH/(NADH + NAD+) and NADPH/(NADPH + NADP+) ratios. Since phosphomannose isomerase is the rate-limiting step for mannose metabolism, our results suggest that mannose impairs erythrocyte defense against oxidants by causing ATP depletion and by impairing the regeneration of reduced pyridine nucleotides by the Embden-Meyerhof and pentose phosphate pathways.
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PMID:Inhibitory effect of mannose on erythrocyte defense against oxidants. 333 78

We studied energy metabolism after experimental subarachnoid hemorrhage in rats. Four different cerebral areas were tested: frontal cortex, occipital cortex, hippocampus, and brainstem. Vmax of the following enzymatic activities was evaluated: in the homogenate: hexokinase, phosphofructokinase, and lactate dehydrogenase for the glycolytic pathway, and glucose-6-phosphate dehydrogenase for the hexose monophosphate shunt; in the purified nonsynaptic mitochondria: NAD+-isocitrate dehydrogenase, citrate synthase, and succinate dehydrogenase for the Krebs cycle, and cytochrome oxidase for the electron transfer chain. We also evaluated some parameters related to the respiration of nonsynaptic mitochondria (State 3, State 4, uncoupled state, respiratory control ratio, and ADP:O ratio). Subarachnoid hemorrhage did not significantly affect Vmax of the enzymatic activities related to anaerobic and aerobic metabolism; however, mitochondrial respiration was affected, particularly in the presence of NADH-producing substrates (glutamate + malate).
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PMID:Bioenergetics of different brain areas after experimental subarachnoid hemorrhage in rats. 335 25

Transient ATP synthesized by preparations enriched with plasmatic membranes of particles from the human placenta in the presence of insulin (4 micrograms/ml) and epidermal growth factor (1 microgram/ml) within 1 min after the addition of hormones at 30 degrees C, was isolated by means of chromatography on Dowex 1 X 8. ATP was synthesized in a medium containing Tris-HCl buffer, pH 7.5, ADP, Mg2+, and Pi during NADH-dependent oxidation in the presence of cytochrome C and oxygen. The amount of ATP was 10(-9) mole/mg protein/min. Quantitative assessment of ATP in lyophilized product was carried out by means of fluorimetry (excitation wavelength--360 nm; emission wavelength--460 nm) of NADH formed during coupled enzymatic reactions involving hexokinase and glucose-6-phosphate dehydrogenase. A possible biological role of peptide growth factor-stimulated formation of transient ATP in plasmatic membranes is discussed.
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PMID:[ATP generation by the plasma membranes of human placental cells as affected by insulin and epidermal growth factor]. 354 74

In basic solutions, pyruvate enolizes and reacts (through its 3-carbon) with the 4-carbon of the nicotinamide ring of NAD+, yielding an NAD-pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the -C(O)NH2 of NAD+ was replaced with -C(S)NH2, -C(O)CH3, and -C(O)H. Of the four, only the last analogue, 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki approximately 1 nM for the A form). Even at high concentrations (20 microM), RAP had little or no effect on rat brain glyceraldehyde-3-phosphate, pyruvate, alpha-ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.
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PMID:Inhibition of lactate production in rat brain extracts and synaptosomes by 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate. 357 4


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