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)

1. The substrate kinetic properties of cerebral hexokinases (mitochondrial and cytoplasmic) were studied at limiting concentrations of both glucose and MgATP(2-). Primary plots of the enzymic activity gave no evidence of a Ping Pong mechanism in three types of mitochondrial preparation tested (intact and osmotically disrupted mitochondria, and the purified mitochondrial enzyme), nor in the purified cytoplasmic preparation. 2. Secondary plots of intercepts from the primary plots (1/v versus 1/s) versus reciprocal of second substrate of the mitochondrial activity gave kinetic constants which differed from those obtained directly from the plots of 1/v versus 1/s or of s/v versus s, although the ratios of the derived constants were consistent. The kinetic constants obtained with the cytoplasmic enzyme from primary and secondary plots were consistent. 3. Deoxyglucose, as alternative substrate, inhibited cytoplasmic hexokinase by competition with glucose, but did not compete when MgATP(2-) was the substrate varied. The K(i) for deoxyglucose when glucose concentrations were varied was 0.25mm. 4. A range of ATP analogues was tested as potential substrates and inhibitors of hexokinase activity. GTP, ITP, CTP, UTP and betagamma-methylene-ATP did not act as substrates, nor did they cause significant inhibition. Deoxy-ATP proved to be almost as effective a substrate as ATP. AMP inhibited but did not act as substrate. 5. N-Acetyl-glucosamine inhibited all preparations competitively when glucose was varied and non-competitively when MgATP(2-) was varied. AMP inhibition was competitive when MgATP(2-) was the substrate varied and non-competitive when glucose was varied. 6. The results are interpreted as providing evidence for a random reaction mechanism in all preparations of brain hexokinase, cytoplasmic and mitochondrial. The kinetic properties and reaction mechanism do not change on extraction and purification of the particulate enzyme. 7. The results are discussed in terms of the participation of hexokinase in regulation of cerebral glycolysis.
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PMID:Cerebral-cortex hexokinase. Elucidation of reaction mechanisms by substrate and dead-end inhibitor kinetic analysis. 512 80

The regulation of extramicrosomal Ca2+ concentration maintained by suspensions of rat insulinoma microsomes was studied using Ca2+-selective minielectrodes. The Ca2+-transporting activity was MgATP dependent and correlated with the endoplasmic reticulum marker NADPH-cytochrome c reductase. When incubated in a high KCl medium containing Mg2+ and phosphate, the microsomes lowered [Ca2+] within less than 10 min to around 0.2 microM. They had a high Ca2+-sequestering activity since they were able to take up and retain several small Ca2+ additions. No evidence for a Na+/Ca2+ countertransport was obtained. The accumulated Ca2+ was released by the Ca2+ ionophore A23187 or upon transforming ATP into ADP using glucose plus hexokinase. The addition of ADP, at concentrations present in cells, resulted in a dose-dependent and reversible net Ca2+ efflux from the microsomes until a higher [Ca2+] steady state was reached. This was specific for ADP since GDP, UDP, CDP, IDP, and the nonhydrolyzable analogue methylene-ADP as well as AMP and cAMP did not reproduce the effect. Insulin secretory granules were unable to lower medium [Ca2+] or to take up a pulse addition of Ca2+. However, most of the large granular calcium content was released by A23187. The addition of Na+ and lowering or increasing medium pH by 0.2 pH unit did not induce Ca2+ uptake or efflux from the secretory granules. The results indicate that insulinoma endoplasmic reticulum but not insulin secretory granules may play a critical role in the regulation of cytosolic Ca2+. A variation in cellular ADP content following secretagogue addition might modulate Ca2+ fluxes across the endoplasmic reticulum and contribute in raising cytosolic Ca2+.
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PMID:Regulation of Ca2+ transport by isolated organelles of a rat insulinoma. Studies with endoplasmic reticulum and secretory granules. 608 82

Glutamine synthetase specific activity increases greater than 100-fold during the insulin-mediated differentiation of confluent 3T3-L1 cells into adipocytes. Incubation of the adipocytes for 22 h with 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline, 0.2 mM 8-bromo-cyclic AMP, 10 micro M epinephrine, or 1 microgram of alpha 1-24 adrenocorticotropic hormone/ml decreased glutamine synthetase by greater than 60%. During the same incubation period, there was no effect of these compounds on protein or on the specific activities of glucose-6-P dehydrogenase or hexokinase. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase activity was half-maximal at 50 micro M dibutyryl cyclic AMP. Furthermore, between 10 micro M and 5 mM dibutyryl cyclic AMP, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase was similar in the absence or presence of 1 microgram of insulin/ml. Immunotitration of glutamine synthetase activity from 3T3 adipocytes indicates that the dibutyryl cyclic AMP-mediated decrease in the activity is due to a decrease in the cellular content of glutamine synthetase molecules. We studied the effects of dibutyryl cyclic AMP on the synthesis and degradation of glutamine synthetase. Synthesis rate was estimated from the incorporation of L-[35S]methionine into glutamine synthetase during a 60-min incubation period. Degradation rate was estimated from the first order disappearance of radioactivity from glutamine synthetase in 3T3 adipocytes previously incubated with L-[35S]methionine. Glutamine synthetase was isolated by immunoprecipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Incubation of 3T3 adipocytes with dibutyrl cyclic AMP resulted in a rapid decline in the apparent synthesis rate of glutamine synthetase. In addition, dibutyryl cyclic AMP treatment increased the initial rate of glutamine synthetase degradation. The half-life of glutamine synthetase was 24.5 h in control cultures and 16 h in dibutyryl cyclic AMP-treated cultures. In contrast, dibutyryl cyclic AMP had little effect on the synthesis or degradation of soluble protein. Our data indicate that the dibutyryl cyclic AMP-mediated decrease in 3T3 adipocyte glutamine synthetase activity results from a decrease in the synthesis rate and an increase in the initial degradation rate of the enzyme.
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PMID:Dibutyryl cyclic AMP decreases glutamine synthetase in cultured 3T3-L1 adipocytes. 610 99

The kinetic properties of the glycosomal hexokinase (HG)2 and phosphofructokinase (PFK) from Trypanosoma brucei bloodstream forms were investigated. Hexokinase has a very high affinity for glucose (Km = 17 microM) and exhibits a broad pH optimum with a maximum at pH 7.8. No indications have been found for regulation of HK activity. Phosphofructokinase behaves as an allosteric protein with respect to its substrate, fructose-6-phosphate. 5'-AMP acts as a positive allosteric effector. The apparent Km for 5'-AMP is extremely low (7 microM). The other substrate for PFK is Mg2+-ATP chelate which activates the enzyme in a hyperbolic manner. Excess of ATP over Mg2+ is inhibitory. The enzyme needs Mg2+ for full activity. Compounds known to be positive or negative heterotrophic modifiers of PFK in other organisms are without effect. It is concluded that PFK and HK probably do not play a regulatory role in glycolysis in T. brucei.
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PMID:Regulation of glycolysis in Trypanosoma brucei: hexokinase and phosphofructokinase activity. 612 64

Glycolysis in Ehrlich ascites tumor cells suspended in buffer containing 5 mM Pi was 50% inhibited by ouabain. In the absence of Pi the inhibition was less striking. Permeabilization of the cells with filipin abolished glycolysis, but glycolysis was restored by addition of Pi and AMP. Neither ouabain nor quercetin inhibited glycolysis in these permeabilized cells. We conclude that quercetin did not inhibit hexokinase sufficiently to affect glycolysis. An extract of Ehrlich ascites tumor cells glycolyzed weakly unless either Pi or an ATPase (e.g. (Na+K+)-ATPase) was added. The low rate of glycolysis of the extract was even further reduced when an endogenous ATPase was removed by precipitation with CaATP. The glycolytic activity of this ATPase-deficient extract was restored by addition of purified (Na+K+)-ATPase or of CaATP-precipitable ATPase. Addition of hexokinase without Pi did not restore glycolytic activity to the extract. An explanation for the contradictory conclusions by Bustamante, E., Morris, H.P., and Pedersen, P.L. (J. Biol. Chem. (1981) 265, 8699-8704) is presented.
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PMID:The role of ATPase in glycolysis of Ehrlich ascites tumor cells. 621 95

Hexokinase (ATP: hexose 6-phosphotransferase, E.C.2.7.1.1) and phosphofructokinase (ATP:fructose-6-phosphate 1-phosphotransferase, E.C.2.7.1.11), two key regulatory enzymes of the glycolytic pathway in vertebrate cells, have been isolated and partially purified from Trypanosoma (Schizotrypanum) cruzi epimastigotes. Both enzymes are associated with particles sedimentable at 105 000 X gav for 1 h and have a high degree of latency; they can be solubilized by sonication. Hexokinase catalyses the phosphorylation of a series of monosaccharides at the following relative rates: D-glucose (100) congruent to D-fructose (97) greater than 2-deoxy-D-glucose (72) congruent to mannose (69) greater than 2-amino-D-glucose (63) greater than 3-O-methyl-D-glucose (21). Very little or no phosphorylating activity was found for D-galactose, N-acetyl-2-amino-D-glucose or 1-alpha-methyl-D-glucose. D-Glucose phosphorylation at fixed ATP concentration follows simple Michaelis-Menten kinetics with Km = 40 microM and Vmax = 440 nmol min-1 mg-1 protein. D-Mannose, 2-deoxy-D-glucose and N-acetyl-2-amino-D-glucose act as competitive inhibitors of glucose phosphorylation, suggesting a single kinase. Mg2+-ATP is the preferred phosphoryl donor, ITP and GTP being much less effective. T. cruzi hexokinase is not inhibited by D-glucose 6-phosphate, or by any of the following compounds (2 mM):D-fructose 6-phosphate, D-fructose 1,6-diphosphate, D-glucose 1,6-diphosphate, phosphoenol pyruvate, L-malate and citrate. Phosphofructokinase displays simple Michaelis-Menten kinetics with no evidence of sigmoidicity with respect to D-fructose 6-phosphate at all ATP concentrations tested, giving a Km of 1.31 mM and Vmax = 400 nmol min-1 mg-1 protein at optimal ATP levels. With respect to ATP, the enzyme exhibits Michaelis-Menten kinetics at low concentration (less than 1 mM) of the substrate (Km = 40 microM at 5 mM MgCl2, pH 7.4). A moderate inhibition is observed at high ATP levels (70% of maximal activity at 2 mM). GTP can substitute for ATP as the phosphoryl donor (Km = 79 microM under the same conditions), but produces only very small inhibitory effects at high concentrations. 5'-AMP activates the enzyme by decreasing its Km with respect to D-fructose 6-phosphate without affecting Vm. Other well-known regulators of the activity of this enzyme in procaryote and vertebrate systems such as citrate, phosphoenol pyruvate, ammonium and phosphate ions have no effect in T. cruzi.
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PMID:Regulation of energy metabolism in Trypanosoma (Schizotrypanum) cruzi epimastigotes. I. Hexokinase and phosphofructokinase. 623 52

The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [3H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high-affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-beta-gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.
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PMID:[3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts. 630 99

Glucagon and dibutyryl cyclic AMP inhibited glucose utilization and lowered fructose 2,6-bisphosphate levels of hepatocytes prepared from fed chickens. Partially purified preparations of chicken liver 6-phosphofructo-1-kinase and fructose 1,6-bisphosphatase were activated and inhibited by fructose 2,6-bisphosphate, respectively. The sensitivities of these enzymes and the changes observed in fructose 2,6-bisphosphate levels are consistent with an important role for this allosteric effector in hormonal regulation of carbohydrate metabolism in chicken liver. In contrast, oleate inhibition of glucose utilization by chicken hepatocytes occurred without change in fructose, 2,6-bisphosphate levels. Likewise, pyruvate inhibition of lactate gluconeogenesis in chicken hepatocytes cannot be explained by changes in fructose 2,6-bisphosphate levels. Exogenous glucose caused a marked increase in fructose 2,6-bisphosphate content of hepatocytes from fasted but not fed birds. Both glucagon and lactate prevented this glucose effect. Fasted chicken hepatocytes responded to lower glucose concentrations than fasted rat hepatocytes, perhaps reflecting the species difference in hexokinase isozymes.
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PMID:Role of fructose 2,6-bisphosphate in the regulation of glycolysis and gluconeogenesis in chicken liver. 631 91

The respiration of rabbit heart mitochondria in the presence of ATP is stimulated by ADP, AMP, creatine and glucose plus hexokinase. The values of V for mitochondrial phosphorylating respiration in the presence of corresponding stimulators are equal to 491 +/- 34, 460 +/- 12, 480 +/- 45 and 463 +/- 72 natoms O2 X min-1 X mg-1 of protein, 37 degrees C. The half-maximal stimulation of respiration is observed at 35 microM AMP, 60 microM ADP and 10 mM creatine in the absence of creatine phosphate. In the presence of creatine phosphate the maximal stimulation of heart mitochondrial respiration is achieved under a combined action of creatine and AMP. The inhibition type of mitochondrial respiration by palmitoyl-CoA depends on the nature of stimulators used. Thus, with ADP or glucose plus hexokinase the inhibition is competitive, while with AMP and creatine an uncompetitive and non-competitive inhibition was observed, respectively. The experimental results are indicative of functional coupling of heart mitochondrial adenylate kinase and creatine phosphokinase with ATP-ADP-translocase. It is assumed that creatine and AMP act as physiological regulators of heart mitochondrial respiration ("feed-back" signals from cytoplasm to mitochondria).
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PMID:[Functional coupling of creatine phosphokinase and adenylate kinase with adenine nucleotide translocase and its role in regulation of heart mitochondrial respiration]. 631 78

We measured hexokinase (EC 2.7.1.1) activity in particulate and soluble fractions isolated from bullfrog (Rana catesbeiana) retinas. Seventy-three percent of the hexokinase (HK) activity was associated with the particulate fraction, 27% with the soluble fraction. Both HK fractions could phosphorylate fructose, glucose, 2-deoxy-D-glucose, and mannose, but not galactose. The Km for glucose was 0.14 mM, for 2-deoxy-D-glucose, 3.6 mM. With glucose as substrate, the Vmax for particulate HK was 125-148 microM retina-1 min-1, for soluble HK, 37 microM retina-1 min-1. Product inhibition of particulate HK activity by glucose 6-phosphate was marked, whereas 2-deoxy-D-glucose 6-phosphate did not inhibit the activity. Cyclic AMP stimulated the HK activity of both retinal fractions nearly twofold at concentrations of 0.2-0.8 mM; AMP was much less effective in this regard.
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PMID:Retinal hexokinase: kinetic properties and the effect of cyclic 3',5'-adenosine monophosphate. 631 79


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