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)

To establish optimum conditions for creatine kinase (EC 2.7.3.2) activity measurement with the creatine phosphate in equilibrium creatine reaction, we re-examined all kinetics factors relevant to an optimal and standardized enzyme assay at 30 and 25 degrees C. We determined the pH optimum in vaious buffers, considering the effect of the type and concentration of the buffer, as well as the influence of various buffer anions on the activity. The relation between activity and substrate concentration was shown and the apparent Michaelis constants of creatine kinase for creatine phosphate and ADP were evaluated. We tested the effect on creatine kinase measurement of the concentration of substrates (glucose and NADP+) in the auxillary and indicator reactions, especially the influence of the added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes on the lag phase, at different temperatures. The NADP+ concentration proved to be the factor limiting the duration of constant reaction rate. We studied the inhibition of creatine kinase and adenylate kinase by AMP and established a convenient AMP concentration. For reactivation of creatine kinase, N-acetyl cysteine as sulfhydryl compound was introduced. Finally, we examined the relationship between activity and temperature.
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PMID:Creatine kinase in serum: 1. Determination of optimum reaction conditions. 0 40

1. The factors influencing the measurement of creatine phosphokinase (CPK) activity in serum by coupled enzymatic methods were investigated to establish optimum conditions for this type of assay. Such a study was indicated following observations by the authors of poor performance of commerically produced reagent kits together with the failure of most of the established an well accepted methods to operate under true optimum zero order kinetics in the reaction phase state. 2. The factors invested were the effects of pH, substrate concentrations (creatine phosphate, glucose and NADP+), added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes, dithiothreitol (DTT) as an activator and conditions of storage of substrate stability. DTT was found to be a suitable activator but not a reactivator of the reaction. The optimum concentrations of creatine phosphate, glucose and NADP+ were found to be 20.0, 20.0 and 2.0 mmol/litre, respectively. Optimum activieies of the enzymes, glucose-6-phosphate dehydrosenase and hexokinase were 1000 and 2000 units/litre, respectively. 3. The between-day precision of the method for measuring serum at pH 6.8 and 30 degrees C at three activity levels under the optimum conditions developed was excellent yielding coefficients of variation ranging from 2.0 to 2.7%.
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PMID:An investigation of factors influencing the measurement of creatine phosphokinase activity in serum using coupled enzymatic methods. 2 6

The enzymes mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, mannitol dehydrogenase and hexokinase participate in an enzymatic cycle in the fungus Alternaria alternata. One turn of the cycle gives the net result: NADH + NADP+ + ATP leads to NAD+ + NADPH + ADP + Pi. The cycle alone can meet the total need of NADPH formation for fat synthesis in the organism. A polyketide producing strain of A. alternata shows a lower mannitol oxidation as well as a lower fat synthesis than a nonproducing mutant, supporting the hypothesis that polyketide formation is favoured at limiting NADPH production. It is further suggested that the mannitol cycle is regulating the glycolytic flux by substrate withdrawal from phosphofructokinase.
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PMID:Production of NADPH in the mannitol cycle and its relation to polyketide formation in Alternaria alternata. 2 47

The denaturation of eight purified yeast enzymes, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, alcohol dehydrogenase, beta-fructosidase, hexokinase and glucose-6-phosphate isomerase, promoted under controlled conditions by the free fatty acids myristic and oleic, is selective. Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1 oxidoreductase, EC 1.1.1.49) is extremely sensitive to destabilization and was studied in greater detail. Results show that chain length and degree of unsaturation of fatty acids are important to their destabilizing effect, and that ligands of the enzyme can afford protection. The denaturation process results in more than one altered form. These results can be viewed in the perspective of the possibility that amphipathic substances, and in particular free fatty acids, may play a role for enzyme degradation in vivo, by initiating steps of selective denaturation.
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PMID:Selective denaturation of several yeast enzymes by free fatty acids. 35 87

In Saccharomyces cerevisiae, a small proportion of the glucose-6-P dehydrogenase activity is firmly associated with the mitochondrial fraction and is not removed by repeated washing or density-gradient centrifugation. However, the enzyme is released by sonic disruption. Mitochondrial glucose-6-P dehydrogenase that is released by sonication and partially purified has been found to be similar to cytosol glucose-6-P dehydrogenase with respect to electrophoretic mobility, isoelectric point, pH optimum, molecular size, and apparent KM's for NADP+ and glucose-6-P. These results indicate that a single species of glucose-6-P dehydrogenase is synthesized in S. cerevisiae and that the enzyme has more than one intracellular location. Mitochondrial glucose-6-P dehydrogenase may be a source of intramitochondrial NADPH and may function with hexokinase and transhydrogenase to provide a pathway for glucose oxidation that is coupled to the synthesis of mitochondrial ATP. A constant proportion of total glucose-6-P dehydrogenase activity remains compartmented in the mitochondrial fraction throughout the growth cycle.
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PMID:Mitochondrial glucose-6-phosphate dehydrogenase from Saccharomyces cerevisiae. 38 93

In this communication the results of applying various histochemical semipermeable membrane techniques to the localization of several enzymes in bovine and porcine heart are presented. The Purkinje fibers of the atrioventricular conducting system of the bovine heart differ from the myocardium proper in containing a greater activity of the glycolytic and gluconeogenetic enzymes--lactate dehydrogenase, glyceraldehyde-phosphate dehydrogenase, hexokinase, glucosephosphate isomerase and phosphoglucomutase, and less activity of the aerobic enzymes--NADH: nitroBT oxidoreductase and isocitrate dehydrogenase (NADP+). The metabolic reactions obtained with Purkinje fibers of the porcine heart are less pronounced. These histochemical findings are in accordance with the impression that Purkinje fibers, compared with the common myocardial fibers, have a higher rate of anaerobic metabolism and a lower rate of aerobic metabolism. The activity of the NADPH regenerating enzymes glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating), and the activity of acid hydrolases such as non-specific esterase and acid phosphatase is higher in the Purkinje fibers of both the bovine and porcine heart.
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PMID:Enzyme histochemical studies on the Purkinje fibers of the atrioventricular system of the bovine and porcine hearts. 66 82

During steady-state, the Pi released in the medium is derived from glucose-6-phosphate which continuously regenerates the ATP hydrolyzed. A membrane potential (delta psi) can be built up in submitochondrial particles using glucose-6-phosphate and hexokinase as an ATP-regenerating system. The energy derived from the membrane potential thus formed, can be used to promote the energy-dependent transhydrogenation from NADH to NADP+ and the uphill electron transfer from succinate to NAD+. In spite of the large differences in the energies of hydrolysis of ATP (delta G degrees = -7.0 to -9.0 kcal/mol) and of glucose-6-phosphate (delta G degrees = -2.5 kcal/mol), the same ratio between Pi production and either NADPH or NADH formation were measured regardless of whether millimolar concentrations of ATP or a mixture of ADP, glucose-6-phosphate and hexokinase were used. Rat liver mitochondria were able to accumulate Ca2+ when incubated in a medium containing hexokinase, ADP and glucose-6-phosphate. The different reaction measured with the use of glucose-6-phosphate and hexokinase were inhibited by glucose concentrations varying from 0.2 to 2 mM. Glucose shifts the equilibrium of the reaction towards glucose-6-phosphate formation thus leading to a decrease of the ATP concentration in the medium.
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PMID:Reversal of oxidative phosphorylation in submitochondrial particles using glucose 6-phosphate and hexokinase as an ATP regenerating system. 149 30

Rats bearing the Walker-256 carcinosarcoma have a profoundly altered liver metabolite content with significant increases in the concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, citrate, lactate, and alanine, while the concentrations of glucose, pyruvate, dihydroxyacetone phosphate, and glutamine are decreased. As a result of these changes both the cytosolic NAD+/NADH ratio and the cytosolic phosphorylation potential are significantly lowered while no changes are detected in either the cytosolic NADP+/NADPH ratio or the mitochondrial NAD+/NADH ratio. These hepatic changes are accompanied by marked increases in the circulating concentrations of lactate, non-esterified fatty acids, and triacylglycerols. The activities of both liver hexokinase and phosphofructokinase are also significantly elevated in the tumor-bearing rats. The changes observed both in the redox state and phosphorylation potential are in agreement with the energy imbalance associated with tumor burden.
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PMID:The energy state of tumor-bearing rats. 199 70

In rat pancreatic islets the effects of cholecystokinin octapeptide (CCK-8) on pentose phosphate shunt (PPS) activity, glucokinase and hexokinase activity, and NADPH, NADP+, NADH, and NAD+ were studied. By elevating the glucose concentration from 3.0 to 8.3 and 16.7 mM the oxidation of [1-14C]- and [6-14C]glucose and the calculated PPS activity were increased in a concentration-dependent manner; 10 nM CCK-8 enhanced selectively the effect on [1-14C]glucose oxidation thereby increasing the PPS activity but only at an intermediate glucose concentration (8.3 mM). CCK-8 had no effect on glucokinase or hexokinase activity and CCK-8 did not influence glucose utilization. By elevating the glucose concentration, total NADPH and NADH were increased and total NADP+ and NAD+ were decreased. CCK-8 (10 nM) increased selectively NADPH and decreased NADP+ but did not change NADH or NAD+; the effect of CCK-8 on NADPH and NADH was only observed in the presence of an intermediate stimulatory glucose concentration (8.3 mM) but not at either a substimulatory glucose concentration or a maximally stimulatory glucose concentration for insulin release (3.0 or 16.7 mM). The data indicate first that CCK-8 does not act on glucose phosphorylation or glucose utilization and second that CCK-8 increases PPS activity and NADPH levels in rat pancreatic islets. Since the concentrations of glucose necessary for these CCK-8 effects are in the range of 8.3 mM and parallel with those necessary for insulin release as shown in earlier observations, glucose oxidation via pentose phosphate shunt and NADPH are suggested to be related to the CCK-8-modulated insulin release.
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PMID:Effect of CCK-8 on pentose phosphate shunt activity, pyridine nucleotides, and glucokinase of rat islets. 264 44

Prolonged intake of low levels of aluminum from the drinking water has been found to increase the aluminum content in rat brain homogenates and to reduce the activity of hexokinase and glucose-6-phosphate dehydrogenase (G6PD). To determine the interaction of G6PD with aluminum in the brain, we have recently purified two isozymes of G6PD (isozymes I and II) from human and pig brain. Unlike isozyme I, isozyme II also had 6-phosphogluconate dehydrogenase (6-PGD) activity. We report here that G6PD isozymes I and II from human and pig brain purified to apparent homogeneity are inactivated by aluminum. Aluminum did not affect the 6-PGD activity of isozyme II. The aluminum-inactivated enzyme contained 1 mol of aluminum/mol of enzyme subunit. The protein-bound metal ion was not dissociated by exhaustive dialysis at 4 degrees C against 10 mM Tris-HCl (pH 7.0) containing 0.2 mM EDTA. Preincubation of aluminum with citrate, NADP+, EDTA, NaF, ATP, and apotransferrin protected the G6PD isozymes against aluminum inactivation. However, when the G6PD isozymes were completely inactivated by aluminum, only citrate, NaF, and apotransferrin restored the enzyme activity. The dissociation constants for the enzyme-aluminum complex of the isozymes varied from 2 to 4 microM, as measured by using NaF, a known chelator for aluminum. Inhibition of G6PD by low levels of aluminum further strengthens the suggested role of aluminum toxicity in the energy metabolism of the brain.
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PMID:Inactivation of glucose-6-phosphate dehydrogenase isozymes from human and pig brain by aluminum. 274 39


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