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)

The purification is described of rat hepatic hexokinase type III and kidney hexokinase type I on a large scale by using a combination of conventional and affinity techniques similar to those previously used for the purification of rat hepatic glucokinase [Holroyde, Allen, Storer, Warsy, Chesher, Trayer, Cornish-Bowden & Walker (1976) Biochem. J. 153, 363-373] and muscle hexokinase type II [Holroyde & Trayer (1976) FEBS Lett. 62, 215-219]. The key to each purification was the use of a Sepharose-N-aminoacylglucosamine affinity matrix in which a high degree of specificity for a particular hexokinase isoenzyme could be introduced by either varying the length of the aminoacyl spacer and/or varying the ligand concentration coupled to the gel. This was predicted from a study of the free solution kinetic properties of the various N-aminoacylglucosamine derivatives used (N-aminopropionyl, N-aminobutyryl, N-aminohexanoyl and N-aminooctanoyl), synthesized as described by Holroyde, Chesher, Trayer & Walker [(1976) Biochem. J. 153, 351-361]. All derivatives were competitive inhibitors, with respect to glucose, of the hexokinase reaction, and there was a direct correlation between the Ki for a particular derivative and its ability to act as an affinity matrix when immobilized to CNBr-activated Sepharose 4B. Muscle hexokinase type II could be chromatographed on the Sepharose conjugates of all four N-aminoacylglucosamine derivatives, although the N-aminohexanoylglucosamine derivative proved best. This same derivative was readily able to bind hepatic glucokinase and hexokinase type III, but Sepharose-N-amino-octanoyl-glucosamine was better for these enzymes and was the only derivative capable of binding kidney hexokinase type I efficiently. Separate studies with yeast hexokinase showed that again only the Sepharose-N-amino-octanoylglucosamine was capable of acting as an efficient affinity matrix for this enzyme. Implications of these studies in our understanding of affinity-chromatography operation are discussed.
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PMID:Purification of the hexokinases by affinity chromatography on sepharose-N-aminoacylglucosamine derivates. Design of affinity matrices from free solution kinetics. 36 63

The adenine nucleotide analog [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) is a potent and highly specific inactivator of yeast 3-phosphoglycerate kinase. Supportive evidence includes the finding that 1) during a 10-min incubation, half-maximal inactivation is given by 10 microM PLP-AMP, 2) covalent incorporation of 1.2 mol of PLP-AMP/mol of enzyme is sufficient to give complete inactivation, and 3) MgATP gives near complete protection against modification and inactivation by PLP-AMP. Following reaction with PLP-AMP and reduction with NaBH4 to form a stable adduct, the enzyme was digested with endoproteinase Lys-C and peptides were separated by reversed-phase high-performance liquid chromatography. The single major labeled peptide was purified and sequenced, and the modified residue was identified as Lys-131. The crystal structure of enzyme in the open conformation shows Lys-131 to reside within a loop of flexible random coil positioned at the outer edge of the central binding cleft, approximately 2 nm from the surface of the cleft that comprises part of the MgATP-binding site (Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kingsman, A. J., and Kingsman, S. M. (1982) EMBO J. 1, 1635-1640). We conclude that the structural element containing Lys-131 undergoes substantial movement during the ligand-induced conformational change known to occur during formation of the ternary complex, resulting in the positioning of a basic residue near a negatively charged substrate. Since similar affinity-labeling results have been presented for hexokinase (Tamura, J. K., LaDine, J. R., and Cross, R. L. (1988) J. Biol. Chem. 263, 7907-7912), we further suggest that movement of positive charge into the central cleft may be a common step in the tight binding of nucleotides by bilobal kinases.
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PMID:The adenine nucleotide-binding site on yeast 3-phosphoglycerate kinase. Affinity labeling of Lys-131 by pyridoxal 5'-diphospho-5'-adenosine. 190 64

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

1. Magnesium ions are the most effective bivalent ions in the glucokinase reaction. 2. The molecular weight of rat hepatic glucokinase is 48000-49000 as assessed by gel filtration on Sephadex G-100. 3. Anomalous kinetic behaviour at low glucose concentrations appears to be due to the formation during the purification procedure of fragments possessing modified catalytic properties, but is unlikely to be of physiological significance. 4. Extension of previous studies (Parry & Walker, 1966) suggests that glucokinase catalyses a reaction of the random Bi Bi type similar to that of yeast hexokinase. 5. The inhibitory effects of various thiol reagents suggest that a thiol group may be involved at or near the binding site of the acceptor molecule.
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PMID:Further properties and possibel mechanism of action of adenosine 5'-triphosphate-D-glucose 6-phosphotransferase from rat liver. 558 91

Key enzyme activities of glycolysis, the pentose-phosphate pathway, the Krebs' cycle and glutaminolysis were measured in lymphocytes obtained from the control (CC), thioglycollate-injected (TG) and Walker 256 tumour-implanted (WT) groups, non-immune and immune inflammatory stimuli, respectively. The rates of incorporation of [2-14C]-thymidine and [5-3H]-uridine into cultured lymphocytes were also determined. The results indicated that the rates of both [2-14C]-thymidine and [5-3H]-uridine incorporation were enhanced in lymphocytes obtained from thioglycollate-injected (by an average of 80 per cent) and tumour-implanted animals (by 2.4-fold) as compared to control rats. Lymphocyte hexokinase activity diminished both in the TG (23 per cent) and WT (61 per cent) groups, whereas glucose 6-phosphate dehydrogenase activity was not altered due to the non-immune inflammatory stimulus, being reduced (23 per cent) in WT rats as compared to CC. The activity of lymphocyte citrate synthase was lowered by thioglycollate (39 per cent) and tumour-implantation (46 per cent). In contrast, glutaminase activity was augmented in lymphocytes from the TG (41 per cent) and was not modified in the WT groups. Taken as a whole, the presence of the Walker 256 tumour did not affect the capacity for glutamine utilization but depressed glucose metabolism in these cells. On the other hand, the non-immune inflammatory stimulus suppressed the activities of glycolysis and the Krebs' cycle and enhanced that of glutaminolysis in lymphocytes.
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PMID:Thioglycollate stimulus modifies lymphocyte metabolism and proliferation. A comparison with lymphocyte activation by Walker 256 tumour implantation. 827 49

It was previously shown that polyunsaturated and saturated fatty acid rich diets affected metabolic and functional changes in macrophages and a variety of immune tissues (thymus, mesenteric lymph nodes and spleen). This study reports metabolic and functional changes in peritoneal macrophages and lymphocytes of Walker-256 ascites cell tumour-bearing rats which were fed (a) normal balanced diet (3% fat), (b) diet enriched (15% fat) with polyunsaturated fatty acids or (c) diet fortified (15% fat) with saturated fatty acids. Neither of the fatty acid enriched diets affected macrophage migration following tumour cell implantation and ascitic cell growth. However both of these fortified fatty acid regimes enhanced the production of H2O2 by macrophages and lymphocytes. The maximum catalytic capacities of hexokinase, glutaminase, glucose-6-phosphate dehydrogenase and glutathione peroxidase were measured in resident and tumour activated macrophages and lymphocytes obtained from rats fed the three fatty acid dietary regimes during seven days of tumour ascites cell growth. Tumour growth caused an increase in the activities of all of the above enzymes in macrophages irrespective of the fatty acid composition of the diet and notably decreased, independent of dietary fatty acid composition, the activities of the enzymes in lymphocytes. Only glutaminase activity in the lymphocytes of tumour bearing animals fed an unsaturated fatty acid-rich diet was not reduced, but was increased by 78%. Moreover macrophages from control rats fed an enriched polyunsaturated fatty acid diet had increased hexokinase activity (21%), decreased glutaminase (48%) and citrate synthase (decreased 41%) relative to the activities of these enzymes in macrophages of animals maintained on a balanced fatty acid diet. The feeding of both fatty acid rich diets did not modify the pattern of lymphocyte responses during the growth of tumour cells in these animals. None of the fatty acid diets modified the growth rate nor the yield of tumour cells in the peritoneal cavity.
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PMID:Effects of various dietary fatty acids on enzyme activities of carbohydrate and glutamine metabolism and the metabolic response of lymphocytes and macrophages during Walker-256 ascites cell tumour growth in rats. 849 May 66

Gamma-linolenic acid (GLA) is known to be an inhibitor of Walker 256 tumour growth in vivo and causes changes in both mitochondrial structure and cellular metabolism. The aim of the present study was to investigate in greater detail the changes in energy metabolism and ultrastructure induced by GLA in this tumour model. A diet containing 5.5% GLA, which is sufficient to cause a 45% decrease in tumour growth, was found to almost double the triacylglycerol (TAG) content of the tumour and to increase the quantity of 20:3 n-6, 20:4 n-6, 22:4 n-6 and 22:5 n-6 in the TAG fraction as determined by gas chromatography-mass spectrometry (GCMS) analysis. Morphometric analysis of the tumour by electron microscopy confirmed this increase in TAG content, identifying a doubling of lipid droplet content in the GLA dietary group. The surface density of mitochondrial cristae was reduced, along with a reduction in the number of contact sites (CS) and matrix granules. These three parameters are likely indicators of a reduction in mitochondrial metabolic activity. Measurement of hexokinase activity identified that much of the total hexokinase activity was in the mitochondrially bound form (66.5%) in the control tumour and that GLA caused a decrease in the amount of enzyme in the bound form (39.3%). The fatty acyl chain composition of the tumour mitochondrial subfractions, outer membranes (OM), CSs and inner membranes (IM) was determined by GCMS. All subfractions showed considerable increases in 20:3 n-6 and decreases in 18:1 n-9, 18:2 n-6 and 22:6 n-3, when exposed to GLA diet. These changes were reflected in a large increase in the n-6/n-3 ratio in the GLA OM vs. the control OM, 21.299 vs. 6.747, respectively. The maximal activity of OM carnitine palmitoyltransferase I (CPT I) was found to be decreased by 61.6% in the GLA diet group. This was accompanied by a decrease in malonyl CoA sensitivity and a decrease in affinity for 16:0 CoA substrate. Such changes in CPT I may be the cause of cytoplasmic acyl CoA accumulation seen in this tumour model. These effects, together with previously reported increases in lipid peroxidation, lead to the conclusion that GLA may cause inhibition of tumour cell growth through separate but interlinked pathways, all of which eventually lead to apoptosis and a decrease in tumour development. The influence of mitochondrial OM fatty acyl chain composition upon two important enzymes of energy metabolism, hexokinase and CPT I, both of which have been linked to apoptosis, is of considerable importance for future studies on fatty acid-induced cell death.
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PMID:Gamma-linolenic acid alters the composition of mitochondrial membrane subfractions, decreases outer mitochondrial membrane binding of hexokinase and alters carnitine palmitoyltransferase I properties in the Walker 256 rat tumour. 1206 51