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)

A study was undertaken to estimate the activities of the key enzymes of glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle in purified rat spermatocytes and spermatids, which have been shown to die in glucose-containing medium and require lactate/pyruvate for maintaining normal ATP concentrations. The aim was to elucidate the changes in the glycolytic and oxidative potential of germ cells undergoing meiosis. Pachytene spermatocytes and round spermatids from adult rat testis were purified to approximately 90% purity by trypsin digestion followed by a combination of centrifugal elutriation and Percoll density gradient centrifugation. After the purity and viability of these cells had been established, their contents of hexokinase, phosphofructokinase, lactate dehydrogenase (LDH) and LDH-X of glycolysis, glucose 6-phosphate dehydrogenase of the pentose phosphate pathway and citrate synthase, aconitase, malate dehydrogenase and 2-oxoglutarate dehydrogenase of the TCA cycle were estimated. These enzymes were also estimated in epididymal spermatozoa for comparison with the testicular germ cells. The results indicate greater activity of glycolytic and pentose phosphate pathway enzymes in spermatocytes than in spermatids, which exhibited greater activity of TCA cycle enzymes than the former. The difference in activity was statistically significant for most of the enzymes studied. In contrast, spermatozoa exhibited markedly greater activity of glycolytic enzymes and significantly lower activity of pentose phosphate pathway and TCA cycle enzymes than did the testicular germ cells. We conclude that the unusual dependence of spermatids exclusively on lactate may be due to their lower glycolytic potential, whereas spermatocytes with comparatively greater glycolytic activity have an intermediate dependence on lactate and are therefore able to utilise lactate, pyruvate, or both, while retaining a better ability to utilise glucose. Spermatozoa with the greatest glycolytic potential and the lowest TCA cycle activity appear to be 'programmed' to utilise exclusively glucose/fructose for energy.
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PMID:Changes in carbohydrate metabolism of testicular germ cells during meiosis in the rat. 953 8

Two techniques for determining enzyme kinetic constants using isothermal titration microcalorimetry are presented. The methods are based on the proportionality between the rate of a reaction and the thermal power (heat/time) generated. (i) An enzyme can be titrated with increasing amounts of substrate, while pseudo-first-order conditions are maintained. (ii) Following a single injection, the change in thermal power as substrate is depleted can be continuously monitored. Both methods allow highly precise kinetic characterization in a single experiment and can be used to measure enzyme inhibition. Applicability is demonstrated using a representative enzyme from each EC classification, including (i) oxidation-reduction activity of DHFR (EC 1.5.1.3); (ii) transferase activity of creatine phosphokinase (EC 2.7.3.2) and hexokinase (EC 2.7.1.1); (iii) hydrolytic activity of Helicobacter pylori urease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.21.16); (iv) lyase activity of heparinase (EC 4.1.1.7); and (v) ligase activity of pyruvate carboxylate (EC 6.4.1.1). This nondestructive method is completely general, enabling precise analysis of reactions in spectroscopically opaque solutions, using physiological substrates. Such a universal assay may have wide applicability in functional genomics.
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PMID:Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? 1155 13

The role of hexokinase in carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of 14CO2 from darkened spinach (Spinacia oleracea), maize (Zea mays) mesophyll, and Chlamydomonas reinhardtii chloroplasts externally supplied with 14C-labeled fructose, glucose, mannose, galactose, maltose, and ribose. Glucose and ribose were the preferred substrates with the Chlamydomonas and maize chloroplasts, respectively. The rate of CO2 release from fructose was about twice that from glucose in the spinach chloroplast. Externally supplied ATP stimulated the rate of CO2 release. The pH optimum for CO2 release was 7.5 with ribose and fructose and 8.5 with glucose as substrates. Probing the outer membrane polypeptides of the intact spinach chloroplast with two proteases, trypsin and thermolysin, decreased 14CO2 release from glucose about 50% but had little effect when fructose was the substrate. Tryptic digestion decreased CO2 release from glucose in the Chlamydomonas chloroplast about 70%. 14CO2 evolution from [1-14C]-glucose-6-phosphate in both chloroplasts was unaffected by treatment with trypsin. Enzymic analysis of the supernatant (stroma) of the lysed spinach chloroplast indicated a hexokinase active primarily with fructose but with some affinity for glucose. The pellet (membranal fraction) contained a hexokinase utilizing both glucose and fructose but with considerably less total activity than the stromal enzyme. Treatment with trypsin and thermolysin eliminated more than 50% of the glucokinase activity but had little effect on fructokinase activity in the spinach chloroplast. Tryptic digestion of the Chlamydomonas chloroplast resulted in a loss of about 90% of glucokinase activity.
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PMID:Respiration of Sugars in Spinach (Spinacia oleracea), Maize (Zea mays), and Chlamydomonas reinhardtii F-60 Chloroplasts with Emphasis on the Hexose Kinases. 1223 48

Intact, multiply protonated proteins of particular mass and charge were selected from ionized protein mixtures and gently landed at different positions on a surface to form a microarray. An array of cytochrome c, lysozyme, insulin, and apomyoglobin was generated, and the deposited proteins showed electrospray ionization mass spectra that matched those of the authentic compounds. Deposited lysozyme and trypsin retained their biological activity. Multiply charged ions of protein kinase A catalytic subunit and hexokinase were also soft-landed into glycerol-based liquid surfaces. These soft-landed kinases phosphorylated LRRASLG oligopeptide and D-fructose, respectively.
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PMID:Preparing protein microarrays by soft-landing of mass-selected ions. 1292 Mar 4

The efflux of lactate dehydrogenase and haemoglobin from human erythrocytes during prolonged incubation at 37 degrees was significantly reduced by ATP, ADP, AMP, UTP, creatine phosphate, or phosphoenolpyruvate and to a lesser extent by fructose, glucose 6-phosphate or fructose 6-phosphate, but not by glucose. Iodoacetate, however, markedly increased the loss of haemoglobin and slightly increased that of lactate dehydrogenase. Phospholipase C greatly accelerated the relase of haemoglobin, lactate dehydrogenase, pyruvate kinase, hexokinase, glucose 6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase from human erythrocytes, but this effect was also reduced in the presence of ATP or ADP. The loss of lactate dehydrogenase, malate dehydrogenase, and pyruvate kinase from the cells treated with phospholipase C increased as their ATP content fell. In a series of experiments in which the action of phospholipase C was stopped by the subsequent addition of trypsin, ATP and ADP (1 mmol/l) significantly reduced the efflux of haemoglobin, but AMP had no such effect. The results are consistent with the conclusion from our previous work that enzyme leakage is related to diminution in the energy content of the cells. The protective action of AMP on cells not treated with phospholipase C, however, differs from earlier findings with rat lymphocytes and it is suggested that in red cells it might be converted into ATP or that it has a direct effect on the permeability of the cell membrane.
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PMID:Factors affecting the release of haemoglobin and enzymes from human erythrocytes. 1563 25

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.
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PMID:Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues. 1700 45

1. A method is described for the isolation of hexokinase from baker's yeast. The method is based mainly on fractionation with alcohol and results See PDF for Structure in a 30-fold increase in specific activity. The final product could be crystallized from ammonium sulfate without change in specific activity. 2. The enzyme catalyzes a transfer of phosphate from adenosinetriphosphate to glucose, fructose, or mannose, the relative rates with these three sugars being 1:1.4:0.3. 3. With glucose as substrate, the turnover number for the crystalline enzyme is 13,000 moles of substrate per 10(5) gm. of protein per minute at 30 degrees and pH 7.5. The temperature coefficient (Q(10 degrees )) between 0 and 30 degrees is 1.9. 4. Magnesium ions are necessary for the activity, the dissociation constant for the Mg(++) -protein complex being 2.6 x 10(-3). Fluoride in concentrations as high as 0.125 M has no inhibitory effect on the enzyme when the Mg(++) and orthophosphate concentrations are 6.5 x 10(-3)M and 1 x 10(-3)M, respectively. 5. The crystalline enzyme shows a loss in activity when highly diluted. This loss in activity can be prevented by diluting in the presence of small amounts of other proteins. Of the various protective proteins tested, insulin was the most effective, providing complete protection in a concentration of 6 micrograms per cc.; with serum albumin, a concentration of 60 micrograms per cc. was necessary. Thiol compounds (cysteine, glutathione) exerted no protective action. 6. The inactivation of the crystalline enzyme on incubation with trypsin can be prevented to a marked degree by the presence of glucose. The instability of crude preparations of yeast hexokinase may be attributed to the presence of proteolytic enzymes, since glucose or fructose has a remarkable protective effect on such preparations.
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PMID:ISOLATION OF HEXOKINASE FROM BAKER'S YEAST. 1987 67

The target proteins of a cytosolic Trx (PfTrx-1) in Plasmodium falciparum with Trx-affinity chromatography were examined. Based on the Trx protein reduction pathway, we generated a cysteine mutant of PfTrx-1, which captures the target protein as a mixed disulfide intermediate. A number of proteins were captured with PfTrx-1(C33S) immobilized on resin and were eluted by DTT treatment. The PfTrx-1(C33S) immobilized resin-captured proteins were trypsin-digested and analyzed on a liquid chromatography-mass spectrometry system. Analysis of the sequence data against databases assigned 20 proteins, four of which had been found previously in P. falciparum, with the remaining 16 being new targets. The potential Trx-target proteins included those in pathways such as the redox cycle, protein biosynthesis, energy metabolism and signal transduction. We captured 4 enzymes in the glycolysis pathway (hexokinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate mutase and L-lactate dehydrogenase (LDH)) as Trx-targets, and we found that PfTrx-1 enhanced the activity of PfGAPDH and PfLDH.
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PMID:Target proteins of the cytosolic thioredoxin in Plasmodium falciparum. 2030 87

This is the first transcriptomic study focusing on immunity in the commercially valuable American lobster (Homarus americanus). We have conducted an in vivo infection trial using the Gram-positive bacterium Aerococcus viridans var. homari to determine how H. americanus responds to this naturally occurring lethal-pathogen. A novel H. americanus microarray was used to measure the transcriptomic changes occurring in over 14,000 genes in the lobster hepatopancreas. Hundreds of new immune genes and isoforms were identified and measured for the first time in this species, and our findings highlight 148 genes of interest involved in H. americanus pathogen response. We verified our microarray results using RT-qPCR on three anti-lipopolysaccharide (ALFHa-1, ALFHa-2, ALFHa-4), a thioredoxin, acute phase serum amyloid protein A, hexokinase and two trypsin genes. RT-qPCR and microarray findings show close agreement and highlight the significant increase in gene expression in many lobster immune genes during A. viridans infection. Differential expression of the ALFHa isoforms may indicate that the H. americanus immune response can be tailored to the class of pathogen causing disease.
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PMID:Differential expression of American lobster (Homarus americanus) immune related genes during infection of Aerococcus viridans var. homari, the causative agent of Gaffkemia. 2322 77

The effect of dietary amylose/amylopectin (AM/AP) ratio on growth, feed utilization, digestive enzyme activities, plasma parameters, and postprandial blood glucose responses was evaluated in juvenile obscure puffer, Takifugu obscurus. Five isonitrogenous (430 g kg(-1) crude protein) and isolipidic (90 g kg(-1) crude lipid) diets containing an equal starch level (250 g kg(-1) starch) with different AM/AP ratio diets of 0/25, 3/22, 6/19, 9/16 and 12/13 were formulated. Each experimental diet was fed to triplicate groups (25 fish per tank), twice daily during a period of 60 days. After the growth trial, a postprandial blood response test was carried out. Fish fed diet 6/19 showed best growth, feed efficiency and protein efficiency ratio. Hepatosomatic index, plasma total cholesterol concentration, liver glycogen and lipid content, and gluconokinase, pyruvate kinase and fructose-1,6-bisphosphatase activities were lower in fish fed highest AM/AP diet (12/13) than in fish fed the low-amylose diets. Activities of liver and intestinal trypsin in fish fed diet 3/22 and diet 6/19 were higher than in fish fed diet 9/16 and diet 12/13. Activities of liver and intestinal amylase and intestinal lipase, and starch digestibility were negatively correlated with dietary AM/AP ratio. Fish fed diet 3/22 and diet 6/19 showed higher plasma total amino acid concentration than fish fed the other diets, while plasma urea nitrogen concentration and activities of alanine aminotransferase and aspartate aminotransferase showed the opposite trend. Equal values were found for viscerosomatic index and condition factor, whole body and muscle composition, plasma high-density and low-density lipoprotein cholesterol concentrations, and activities of lipase and hexokinase and glucose-6-phosphatase in liver. Postprandial plasma glucose and triglyceride peak value of fish fed diet 12/13 were lower than in fish fed the low-amylose diets, and the peak time of plasma glucose was later than in fish fed the other diets. Plasma glucose and triglyceride concentrations showed a significant difference at 2 and 4 h after a meal and varied between dietary treatments. According to regression analysis of weight gain against dietary AM/AP ratio, the optimum dietary AM/AP ratio for maximum growth of obscure puffer was 0.25. The present result indicates that dietary AM/AP ratio could affect growth performance and feed utilization, some plasma parameters, digestive enzyme as well as hepatic glucose metabolic enzyme activities in juvenile obscure puffer.
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PMID:Effects of dietary amylose/amylopectin ratio on growth performance, feed utilization, digestive enzymes, and postprandial metabolic responses in juvenile obscure puffer Takifugu obscurus. 2471 Jun 1


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