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1
Radojković, J. y T. Ureta. "Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C". Biochemical Journal 242, n.º 3 (15 de marzo de 1987): 895–903. http://dx.doi.org/10.1042/bj2420895.
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The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.
2
Magnani, M., G. Serafini y V. Stocchi. "Hexokinase type I multiplicity in human erythrocytes". Biochemical Journal 254, n.º 2 (1 de septiembre de 1988): 617–20. http://dx.doi.org/10.1042/bj2540617.
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Hexokinase I in human erythrocytes exists in multiple molecular forms that differ in isoelectric points. By means of Western blotting and immunodetection of total glucose-phosphorylating activity by using an antibody raised in rabbit against homogeneous human placenta hexokinase I, a single protein band was detected. Identical results were also obtained by immunoaffinity chromatography of the partially purified enzyme. Separation of the three major hexokinase I subtypes (Ia, Ib and Ic) by h.p.l.c. ion-exchange chromatography and immunodetection following electrophoretic blotting confirmed that each hexokinase subtype showed the same apparent Mr of 112,000, which is the value obtained for the high-Mr hexokinase I from human placenta. Purification of erythrocyte hexokinase by a combination of several procedures including dye-ligand and affinity chromatography that were previously successfully applied to the purification of other mammalian hexokinases type I produced a 35,000-fold-purified enzyme that showed several contaminants after SDS/polyacrylamide-gel electrophoresis. Only one of these peptides was found to be recognized by anti-(hexokinase I) IgG, suggesting that proteolytic degradation does not occur and that hexokinases Ia, Ib and Ic have the same apparent Mr.
3
Vischer, U., B. Blondel, C. B. Wollheim, W. Höppner, H. J. Seitz y P. B. Iynedjian. "Hexokinase isoenzymes of RIN-m5F insulinoma cells. Expression of glucokinase gene in insulin-producing cells". Biochemical Journal 241, n.º 1 (1 de enero de 1987): 249–55. http://dx.doi.org/10.1042/bj2410249.
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We have analysed the pattern of expression of the hexokinase isoenzyme group in RIN-m5F insulinoma cells. Three hexokinase forms were resolved by DEAE-cellulose chromatography. The most abundant isoenzyme co-eluted with hexokinase type II from rat adipose tissue and displayed a Km for glucose of 0.15 mM, similar to the adipose-tissue enzyme. Hexokinase type II was in large part associated with a particulate subcellular fraction in RIN-m5F cells. The two other hexokinases separated by ion-exchange chromatography were an enzyme similar to hexokinase type I from brain and glucokinase (or hexokinase type IV). The latter isoenzyme was identified as the liver-type glucokinase by the following properties: co-elution with hepatic glucokinase from DEAE-cellulose and DEAE-Sephadex; sigmoid saturation kinetics with glucose with half-maximal velocity at 5.6 mM and Hill coefficient (h) of 1.54; suppression of enzyme activity by antibodies raised against rat liver glucokinase; apparent Mr of 56,500 and pI of 5.6, as shown by immunoblotting after one- and two-dimensional gel electrophoresis; peptide map identical with that of hepatic glucokinase after proteolysis with chymotrypsin and papain. These data indicate that the gene coding for hepatic glucokinase is expressed in RIN-m5F cells, a finding consistent with indirect evidence for the presence of glucokinase in the beta-cell of the islet of Langerhans. On the other hand, the overall pattern of hexokinases is distinctly different in RIN-m5F cells and islets of Langerhans, since hexokinase type II appears to be lacking in islets. Alteration in hexokinase expression after tumoral transformation has been reported in other systems.
4
van Wijk, Richard, Gert Rijksen, Eric G. Huizinga, Hendrik K. Nieuwenhuis y Wouter W. van Solinge. "HK Utrecht: missense mutation in the active site of human hexokinase associated with hexokinase deficiency and severe nonspherocytic hemolytic anemia". Blood 101, n.º 1 (1 de enero de 2003): 345–47. http://dx.doi.org/10.1182/blood-2002-06-1851.
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Abstract Hexokinase deficiency is a rare autosomal recessive disease with a clinical phenotype of severe hemolysis. We report a novel homozygous missense mutation in exon 15 (c.2039C>G, HK [hexokinase] Utrecht) of HK1, the gene that encodes red blood cell–specific hexokinase-R, in a patient previously diagnosed with hexokinase deficiency. The Thr680Ser substitution predicted by this mutation affects a highly conserved residue in the enzyme's active site that interacts with phosphate moieties of adenosine diphosphate, adenosine triphosphate (ATP), and inhibitor glucose-6-phosphate. We correlated the molecular data to the severe clinical phenotype of the patient by means of altered enzymatic properties of partially purified hexokinase from the patient, notably with respect to Mg2+-ATP binding. These kinetic properties contradict those obtained from a recombinant mutant brain hexokinase-I with the same Thr680Ser substitution. This contradiction thereby stresses the valuable contribution of studying patients with hexokinase deficiency to achieve a better understanding of hexokinase's key role in glycolysis.
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Bergmanni, Fritz y Leon Mejnartowicz. "Substrate specificity of glucokinase and fructokinase of several conifer species". Acta Societatis Botanicorum Poloniae 71, n.º 2 (2014): 125–27. http://dx.doi.org/10.5586/asbp.2002.014.
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Annual plant species were found to have several distinct classes of hexokinases which are specific for different hexoses, such as glucose, fructose and mannose. In conifers one isozyme of hexokinase could be found in genetic studies if only glucose was employed as substrate. If fructose was substituted for glucose, another isozyme zone different from the common hexokinase could be observed in zymograms of extracts from seed tissues of Norway spruce, Scots pine and silver fir. Hence these three conifer species possess at least two different hexokinases, glucokinase and fructokinase.
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Galina, A., M. Reis, M. C. Albuquerque, A. G. Puyou, M. T. G. Puyou y L. de Meis. "Different properties of the mitochondrial and cytosolic hexokinases in maize roots". Biochemical Journal 309, n.º 1 (1 de julio de 1995): 105–12. http://dx.doi.org/10.1042/bj3090105.
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After tissue homogenization, 43% of the total hexokinase activity found in maize radicles was recovered in the mitochondrial fraction and 35% was soluble, in the cytosol. The maize submitochondrial particles obtained after mitochondrial sonication retained a high hexokinase activity. The mitochondrial respiration (state 4 rate) was activated by glucose. This activation was blocked by carboxyatractyloside (0.5 mM) and by oligomycin (2 micrograms/ml). The affinities for ATP and glucose of both soluble and membrane-bound maize hexokinases are similar to those of yeast hexokinase. The Km for ATP of these different forms of hexokinase varied between 0.15 and 0.37 mM, and the Km for glucose between 0.05 and 0.13 mM. A major difference between the two maize hexokinase forms is that only the mitochondrial enzyme was strongly inhibited by ADP (Ki 0.04 mM). The soluble forms of hexokinase found both in the cytosol of maize radicles and in yeast are not inhibited by ADP. In a previous report [de Meis, Grieco and Galina (1992) FEBS Lett. 308, 197-201] it was shown that the mitochondrial F1-F0-ATPase can use glucose 6-phosphate and yeast hexokinase as an ATP regenerating system. We now show that the membrane-bound hexokinase and glucose 6-phosphate can also serve as an ATP regenerating system for the mitochondria of maize radicles provided that the ADP concentration is kept below 0.05 mM. Higher ADP concentrations inhibit the reverse reaction of the mitochondrial hexokinase.
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Victorovich, Khrustalev Vladislav, Lelevich Sergey Vladimirovich y Barkovsky Eugene Victorovich. "Zebra Finch Glucokinase Containing Two Homologous Halves Is an In Silico Chimera". ISRN Computational Biology 2013 (7 de noviembre de 2013): 1–6. http://dx.doi.org/10.1155/2013/790240.
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Chimerical nature of the gene annotated as Zebra finch (Taeniopygia guttata) glucokinase (hexokinase IV) has been proved in this study. N-half of the protein encoded by that gene shows similarity with glucokinase from other vertebrates, while its C-half shows similarity with C-halves of hexokinases II. We mapped 7 new exons coding for N-half of hexokinase II and 4 new exons coding for glucokinase of Zebra finch. Finally, we reconstructed normal genes coding for Zebra finch glucokinase and hexokinase II which are situated in “head-to-tail” orientation on the chromosome 22. Because of the error in gene annotation, exons encoding N-half of normal glucokinase have been fused with exons encoding C-half of normal hexokinase II, even though they are separated from each other by the sequence 98066 nucleotides in length.
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Magnani, M., M. Bianchi, A. Casabianca, V. Stocchi, A. Daniele, F. Altruda, M. Ferrone y L. Silengo. "A recombinant human ‘mini’-hexokinase is catalytically active and regulated by hexose 6-phosphates". Biochemical Journal 285, n.º 1 (1 de julio de 1992): 193–99. http://dx.doi.org/10.1042/bj2850193.
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Mammalian hexokinase type I is a 100 kDa enzyme that has been considered to be evolved from an ancestral 50 kDa yeast-type hexokinase, insensitive to product inhibition, by gene duplication and fusion. According to this model, and based on many experimental data, the catalytic site is associated with the C-terminal half of the enzyme, although an allosteric site for the binding of glucose 6-phosphate could be present on the N-terminal half of the molecule. We have isolated a cDNA clone of hexokinase from a lambda gt11 human placenta library comprising 2658 bp, containing a single open reading frame of 1893 nucleotides, which encodes a truncate form of hexokinase starting from asparagine-287 to the terminal serine-917. This clone was further digested with restriction enzyme NcoI to obtain almost only the C-terminal half of human hexokinase starting from methionine-455 to the terminal amino acid and was overexpressed in active form in Escherichia coli and purified by ion-exchange h.p.l.c. The overexpressed ‘mini’-hexokinase was found not only to catalyse glucose phosphorylation, but also to be inhibited by glucose 6-phosphate and other mono- and bis-phosphate sugars exactly like the complete mammalian enzyme. These results suggest that the C-terminal half of human hexokinase, in addition to the catalytic site, also contains the regulatory site and that the evolutionary relationship between the hexokinases should be reconsidered by including the appearance of a regulatory site before the gene duplication.
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Wasserman, David H. "Insulin, Muscle Glucose Uptake, and Hexokinase: Revisiting the Road Not Taken". Physiology 37, n.º 3 (1 de mayo de 2022): 115–27. http://dx.doi.org/10.1152/physiol.00034.2021.
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Research conducted over the last 50 yr has provided insight into the mechanisms by which insulin stimulates glucose transport across the skeletal muscle cell membrane Transport alone, however, does not result in net glucose uptake as free glucose equilibrates across the cell membrane and is not metabolized. Glucose uptake requires that glucose is phosphorylated by hexokinases. Phosphorylated glucose cannot leave the cell and is the substrate for metabolism. It is indisputable that glucose phosphorylation is essential for glucose uptake. Major advances have been made in defining the regulation of the insulin-stimulated glucose transporter (GLUT4) in skeletal muscle. By contrast, the insulin-regulated hexokinase (hexokinase II) parallels Robert Frost’s “The Road Not Taken.” Here the case is made that an understanding of glucose phosphorylation by hexokinase II is necessary to define the regulation of skeletal muscle glucose uptake in health and insulin resistance. Results of studies from different physiological disciplines that have elegantly described how hexokinase II can be regulated are summarized to provide a framework for potential application to skeletal muscle. Mechanisms by which hexokinase II is regulated in skeletal muscle await rigorous examination.
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Khan, Md Wasim, Xianzhong Ding, Scott J. Cotler, Michael Clarke y Brian T. Layden. "Studies on the Tissue Localization of HKDC1, a Putative Novel Fifth Hexokinase, in Humans". Journal of Histochemistry & Cytochemistry 66, n.º 5 (5 de febrero de 2018): 385–92. http://dx.doi.org/10.1369/0022155418756849.
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Hexokinase domain component 1 (HKDC1) is a recently discovered novel protein, which is being promoted as a putative fifth hexokinase. Although the exact role HKDC1 plays in physiology is still unclear, it has been shown to be important during pregnancy in the regulation of glucose homeostasis. In this study, we have comprehensively studied the expression pattern of HKDC1 in the human body. Using human tissue sample, immunohistochemistry imaging was performed. Our studies indicate that the tissues with highest HKDC1 expression were the brush border epithelium of the intestines, parts of the pancreas, and lung alveolar macrophages. Future directions will be to understand the role of this fifth hexokinase in these tissues, with a focus on its relative function as compared with other endogenously expressed hexokinases.
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Agius, L. "Hexokinase and glucokinase binding in permeabilized guinea-pig hepatocytes". Biochemical Journal 303, n.º 3 (1 de noviembre de 1994): 841–46. http://dx.doi.org/10.1042/bj3030841.
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The release of glucokinase (hexokinase IV) from digitonin-permeabilized hepatocytes from rat, guinea pig or mouse liver is inhibited by physiological concentrations of Mg2+ (> 0.25 mM). Preincubation of hepatocytes with fructose increases glucokinase release during permeabilization in the presence of Mg2+ but decreases glucokinase release in the absence of Mg2+, suggesting that fructose causes translocation of glucokinase from the Mg(2+)-dependent site. Glucose (25 mM) and sorbitol (1 mM) also induce translocation of glucokinase from the Mg(2+)-dependent site in guinea-pig, as in rat hepatocytes, but glucose is less effective than fructose or sorbitol, and the concentrations of fructose and sorbitol that cause half-maximal activation (A50) are 3-fold and 20-fold higher, respectively, in guinea-pig than in rat hepatocytes (170 microM and 257 microM, compared with 61 microM and 13 microM). Dihydroxyacetone and glycerol have no effect on fructose-induced or sorbitol-induced translocation in guinea-pig hepatocytes, in contrast with the potentiation and inhibition, respectively, by these substrates in rat hepatocytes. Some, but not all, of the differences between rat and guinea-pig hepatocytes could be due to the more reduced cytoplasmic NADH/NAD+ redox state in guinea-pig cells. The activity of low-Km hexokinases accounts for 30% of total hexokinase activity (low-Km hexokinases + glucokinase) in guinea-pig hepatocytes. Of the low-Km hexokinase activity, approx. 30% is released in the presence of Mg2+, 9% shows Mg(2+)-dependent binding and 60% shows Mg(2+)-independent binding. There was no substrate-induced translocation of low-Km hexokinase activity, indicating that translocation is specific for hexokinase IV.
12
Kharitonov, S., A. Zikiriahodzhaev, M. Ermoshchenkova, A. Sukhot’ko, M. Fedorova, E. Pudova, B. Alekseev, A. Kaprin y A. Kudryavtseva. "HEXOKINASES IN BREAST CANCER". International Journal of Biosciences and Biotechnology 4, n.º 2 (2 de abril de 2017): 110. http://dx.doi.org/10.24843/ijbb.2017.v04.i02.p05.
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Hexokinases are one of the key enzymes involved in the process of glycolysis. The level of expression of hexokinases is widely studied in breast cancer as a possible marker of unfavorable prognosis and aggressiveness of tumors. The level of expression of hexokinase may reflect the level of glycolysis activation and, thus, indicate samples with the most altered cellular metabolism.
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Ma, H. y D. Botstein. "Effects of null mutations in the hexokinase genes of Saccharomyces cerevisiae on catabolite repression". Molecular and Cellular Biology 6, n.º 11 (noviembre de 1986): 4046–52. http://dx.doi.org/10.1128/mcb.6.11.4046-4052.1986.
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Saccharomyces cerevisiae has two homologous hexokinases, I and II; they are 78% identical at the amino acid level. Either enzyme allows yeast cells to ferment fructose. Mutant strains without any hexokinase can still grow on glucose by using a third enzyme, glucokinase. Hexokinase II has been implicated in the control of catabolite repression in yeasts. We constructed null mutations in both hexokinase genes, HXK1 and HXK2, and studied their effect on the fermentation of fructose and on catabolite repression of three different genes in yeasts: SUC2, CYC1, and GAL10. The results indicate that hxk1 or hxk2 single null mutants can ferment fructose but that hxk1 hxk2 double mutants cannot. The hxk2 single mutant, as well as the double mutant, failed to show catabolite repression in all three systems, while the hxk1 null mutation had little or no effect on catabolite repression.
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Srivastava, Shanti Swaroop, Joseph E. Darling, Jimmy Suryadi, James C. Morris, Mark E. Drew y Sriram Subramaniam. "Plasmodium vivax and human hexokinases share similar active sites but display distinct quaternary architectures". IUCrJ 7, n.º 3 (26 de marzo de 2020): 453–61. http://dx.doi.org/10.1107/s2052252520002456.
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Malaria is a devastating disease caused by a protozoan parasite. It affects over 300 million individuals and results in over 400 000 deaths annually, most of whom are young children under the age of five. Hexokinase, the first enzyme in glucose metabolism, plays an important role in the infection process and represents a promising target for therapeutic intervention. Here, cryo-EM structures of two conformational states of Plasmodium vivax hexokinase (PvHK) are reported at resolutions of ∼3 Å. It is shown that unlike other known hexokinase structures, PvHK displays a unique tetrameric organization (∼220 kDa) that can exist in either open or closed quaternary conformational states. Despite the resemblance of the active site of PvHK to its mammalian counterparts, this tetrameric organization is distinct from that of human hexokinases, providing a foundation for the structure-guided design of parasite-selective antimalarial drugs.
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Ma, H. y D. Botstein. "Effects of null mutations in the hexokinase genes of Saccharomyces cerevisiae on catabolite repression." Molecular and Cellular Biology 6, n.º 11 (noviembre de 1986): 4046–52. http://dx.doi.org/10.1128/mcb.6.11.4046.
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Saccharomyces cerevisiae has two homologous hexokinases, I and II; they are 78% identical at the amino acid level. Either enzyme allows yeast cells to ferment fructose. Mutant strains without any hexokinase can still grow on glucose by using a third enzyme, glucokinase. Hexokinase II has been implicated in the control of catabolite repression in yeasts. We constructed null mutations in both hexokinase genes, HXK1 and HXK2, and studied their effect on the fermentation of fructose and on catabolite repression of three different genes in yeasts: SUC2, CYC1, and GAL10. The results indicate that hxk1 or hxk2 single null mutants can ferment fructose but that hxk1 hxk2 double mutants cannot. The hxk2 single mutant, as well as the double mutant, failed to show catabolite repression in all three systems, while the hxk1 null mutation had little or no effect on catabolite repression.
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Schoeniger, Axel, Philipp Wolf y Frank Edlich. "How Do Hexokinases Inhibit Receptor-Mediated Apoptosis?" Biology 11, n.º 3 (8 de marzo de 2022): 412. http://dx.doi.org/10.3390/biology11030412.
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The regulated cell death apoptosis enables redundant or compromised cells in ontogeny and homeostasis to remove themselves receptor-dependent after extrinsic signaling or after internal stress by BCL-2 proteins on the outer mitochondrial membrane (OMM). Mitochondrial BCL-2 proteins are also often needed for receptor-mediated signaling in apoptosis. Then, the truncated BH3-only protein BID (tBID) blocks retrotranslocation of the pro-apoptotic BCL-2 proteins BAX and BAK from the mitochondria into the cytosol. BAX and BAK in turn permeabilize the OMM. Although the BCL-2 proteins are controlled by a complex regulatory network, a specific mechanism for the inhibition of tBID remained unknown. Curiously, it was suggested that hexokinases, which channel glucose into the metabolism, have an intriguing function in the regulation of apoptosis. Recent analysis of transient hexokinase interactions with BAX revealed its participation in the inhibition of BAX and also BAK by retrotranslocation from mitochondria to the cytosol. In contrast to general apoptosis inhibition by anti-apoptotic BCL-2 proteins, hexokinase I and hexokinase 2 specifically inhibit tBID and thus the mitochondrial apoptosis pathway in response to death receptor signaling. Mitochondrial hexokinase localization and BH3 binding of cytosolic hexokinase domains are prerequisites for protection against receptor-mediated cell death, whereas glucose metabolism is not. This mechanism protects cells from apoptosis induced by cytotoxic T cells.
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Prior, C., P. Mamessier, H. Fukuhara, X. J. Chen y M. Wesolowski-Louvel. "The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis". Molecular and Cellular Biology 13, n.º 7 (julio de 1993): 3882–89. http://dx.doi.org/10.1128/mcb.13.7.3882-3889.1993.
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The RAG1 gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAG5, and RAG8 genes are trans-acting genes controlling the expression of the RAG1 gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAG5 gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. rag5 mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high- and low-affinity transport systems of glucose were affected in rag5 mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG1 gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the HXK2 gene of S. cerevisiae and complementation of hxk1 hxk2 mutations of S. cerevisiae by the RAG5 gene showed that RAG5 and HXK2 were equivalent for sugar-phosphorylating activity but that RAG5 could not restore glucose repression in the S. cerevisiae hexokinase mutants.
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Prior, C., P. Mamessier, H. Fukuhara, X. J. Chen y M. Wesolowski-Louvel. "The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis." Molecular and Cellular Biology 13, n.º 7 (julio de 1993): 3882–89. http://dx.doi.org/10.1128/mcb.13.7.3882.
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The RAG1 gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAG5, and RAG8 genes are trans-acting genes controlling the expression of the RAG1 gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAG5 gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. rag5 mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high- and low-affinity transport systems of glucose were affected in rag5 mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG1 gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the HXK2 gene of S. cerevisiae and complementation of hxk1 hxk2 mutations of S. cerevisiae by the RAG5 gene showed that RAG5 and HXK2 were equivalent for sugar-phosphorylating activity but that RAG5 could not restore glucose repression in the S. cerevisiae hexokinase mutants.
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Deeb, S. S., M. Malkki y M. Laakso. "Human Hexokinase II: Sequence and Homology to Other Hexokinases". Biochemical and Biophysical Research Communications 197, n.º 1 (noviembre de 1993): 68–74. http://dx.doi.org/10.1006/bbrc.1993.2442.
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Majewski, Nathan, Veronique Nogueira, R. Brooks Robey y Nissim Hay. "Akt Inhibits Apoptosis Downstream of BID Cleavage via a Glucose-Dependent Mechanism Involving Mitochondrial Hexokinases". Molecular and Cellular Biology 24, n.º 2 (15 de enero de 2004): 730–40. http://dx.doi.org/10.1128/mcb.24.2.730-740.2004.
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ABSTRACT The serine/threonine kinase Akt/protein kinase B inhibits apoptosis induced by a variety of stimuli, including overexpression or activation of proapoptotic Bcl-2 family members. The precise mechanisms by which Akt prevents apoptosis are not completely understood, but Akt may function to maintain mitochondrial integrity, thereby preventing cytochrome c release following an apoptotic insult. This effect may be mediated, in part, via promotion of physical and functional interactions between mitochondria and hexokinases. Here we show that growth factor deprivation induced proteolytic cleavage of the proapoptotic Bcl-2 family member BID to yield its active truncated form, tBID. Activated Akt inhibited mitochondrial cytochrome c release and apoptosis following BID cleavage. Akt also antagonized tBID-mediated BAX activation and mitochondrial BAK oligomerization, two downstream events thought to be critical for tBID-induced apoptosis. Glucose deprivation, which impaired the ability of Akt to maintain mitochondrion-hexokinase association, prevented Akt from inhibiting BID-mediated apoptosis. Interestingly, tBID independently elicited dissociation of hexokinases from mitochondria, an effect that was antagonized by activated Akt. Ectopic expression of the amino-terminal half of hexokinase II, which is catalytically active and contains the mitochondrion-binding domain, consistently antagonized tBID-induced apoptosis. These results suggest that Akt inhibits BID-mediated apoptosis downstream of BID cleavage via promotion of mitochondrial hexokinase association and antagonism of tBID-mediated BAX and BAK activation at the mitochondria.
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Fleck, Christian B. y Matthias Brock. "Aspergillus fumigatus Catalytic Glucokinase and Hexokinase: Expression Analysis and Importance for Germination, Growth, and Conidiation". Eukaryotic Cell 9, n.º 7 (7 de mayo de 2010): 1120–35. http://dx.doi.org/10.1128/ec.00362-09.
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ABSTRACT Fungi contain several hexokinases, which are involved either in sugar phosphorylation or in carbon source sensing. Glucose and fructose phosphorylations appear to rely exclusively on glucokinase and hexokinase. Here, we characterized the catalytic glucokinase and hexokinase from the opportunistic human pathogen Aspergillus fumigatus and showed that both enzymes display different biochemical properties and play different roles during growth and development. Glucokinase efficiently activates glucose and mannose but activates fructose only to a minor extent. Hexokinase showed a high efficiency for fructose activation but also activated glucose and mannose. Transcript and activity determinations revealed high levels of glucokinase in resting conidia, whereas hexokinase was associated mainly with the mycelium. Consequentially, a glucokinase mutant showed delayed germination at low glucose concentrations, whereas colony growth was not overly affected. The deletion of hexokinase had only a minor impact on germination but reduced colony growth, especially on sugar-containing media. Transcript determinations from infected mouse lungs revealed the expression of both genes, indicating a contribution to virulence. Interestingly, a double-deletion mutant showed impaired growth not only on sugars but also on nonfermentable nutrients, and growth on gluconeogenic carbon sources was strongly suppressed in the presence of glucose. Furthermore, the glkA hxkA deletion affected cell wall integrity, implying that both enzymes contribute to the cell wall composition. Additionally, the absence of either enzyme deregulated carbon catabolite repression since mutants displayed an induction of isocitrate lyase activity during growth on glucose-ethanol medium. Therefore, both enzymes seem to be required for balancing carbon flux in A. fumigatus and are indispensable for growth under all nutritional conditions.
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Bearham, Jade, James P. Garnett, Victoria Schroeder, Matthew G. S. Biggart y Deborah L. Baines. "Effective glucose metabolism maintains low intracellular glucose in airway epithelial cells after exposure to hyperglycemia". American Journal of Physiology-Cell Physiology 317, n.º 5 (1 de noviembre de 2019): C983—C992. http://dx.doi.org/10.1152/ajpcell.00193.2019.
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The airway epithelium maintains differential glucose concentrations between the airway surface liquid (ASL, ~0.4 mM) and the blood/interstitium (5–6 mM), which is important for defense against infection. Glucose primarily moves from the blood to the ASL via paracellular movement, down its concentration gradient, across the tight junctions. However, there is evidence that glucose can move transcellularly across epithelial cells. Using a Förster resonance energy transfer sensor for glucose, we investigated intracellular glucose concentrations in airway epithelial cells and the role of hexokinases in regulating intracellular glucose concentrations in normoglycemic and hyperglycemic conditions. Our findings indicated that in airway epithelial cells (H441 or primary human bronchial epithelial cells) exposed to 5 mM glucose (normoglycemia), intracellular glucose concentration is in the micromolar range. Inhibition of facilitative glucose transporters (GLUTs) with cytochalasin B reduced intracellular glucose concentration. When cells were exposed to 15 mM glucose (hyperglycemia), intracellular glucose concentration was reduced. Airway cells expressed hexokinases I, II, and III. Inhibition with 3-bromopyruvate decreased hexokinase activity by 25% and elevated intracellular glucose concentration, but levels remained in the micromolar range. Exposure to hyperglycemia increased glycolysis, glycogen, and sorbitol. Thus, glucose enters the airway cell via GLUTs and is then rapidly processed by hexokinase-dependent and hexokinase-independent metabolic pathways to maintain low intracellular glucose concentrations. We propose that this prevents transcellular transport and aids the removal of glucose from the ASL and that the main route of entry for glucose into the ASL is via the paracellular pathway.
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Stachyra-Strawa, Paulina, Paweł Cisek, Michał Janiszewski y Ludmiła Grzybowska-Szatkowska. "The role of hexokinase in cancer". Postępy Higieny i Medycyny Doświadczalnej 74 (22 de mayo de 2020): 144–50. http://dx.doi.org/10.5604/01.3001.0014.1528.
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A thorough understanding of the processes occurring in cancer cells is necessary to make cancer treatment as effective as possible. Changes in cellular metabolism in relation to normal cells are considered particularly important. One of the most interesting and promising areas is glucose metabolism and the factors affecting this process, with special emphasis on the potential role of hexokinases, especially the isoform II of this enzyme. Hexokinases (HK) are transferase enzymes involved in the process of glycolysis. Hexokinase II (HK II) plays an important role in initiating and maintaining the glycolysis process at a high level of efficiency, which is crucial for the growth and proliferation of cancer cells. An increase in the number of copies of the HK II gene and increased transcription of this enzyme resulting in the suppression of apoptosis and the enhancement of cell proliferation have been found in tumor cells. Hexokinase II also participates in the Crabtree effect by affecting the amount of ATP and thus the efficiency of the Ca2+ removal process outside the cell membrane by Ca2+ ATPase. Overexpression of HK II has thus far been found in pancreatic cancer, gastric cancer, breast cancer, squamous cell carcinoma of the larynx, glioblastoma multiforme, ovarian cancer and biliary tract cancer, indicating the possible key role of this enzyme in their formation and progression and providing the basis for seeking potential benefits of cancer treatment using HK II as a target of new drugs.
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Walsh, R. B., D. Clifton, J. Horak y D. G. Fraenkel. "Saccharomyces cerevisiae null mutants in glucose phosphorylation: metabolism and invertase expression." Genetics 128, n.º 3 (1 de julio de 1991): 521–27. http://dx.doi.org/10.1093/genetics/128.3.521.
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Abstract A congenic series of Saccharomyces cerevisiae strains has been constructed which carry, in all combinations, null mutations in the three genes for glucose phosphorylation: HXK1, HXK2 and GLK1, coding hexokinase 1 (also called PI or A), hexokinase 2 (PII or B), and glucokinase, respectively: i.e., eight strains, all of which grow on glucose except for the triple mutant. All or several of the strains were characterized in their steady state batch growth with 0.2% or 2% glucose, in aerobic as well as respiration-inhibited conditions, with respect to growth rate, yield, and ethanol formation. Glucose flux values were generally similar for different strains and conditions, provided they contained either hexokinase 1 or hexokinase 2. And their aerobic growth, as known for wild type, was largely fermentative with ca. 1.5 mol ethanol made per mol glucose used. The strain lacking both hexokinases and containing glucokinase was an exception in having reduced flux, a result fitting with its maximal rate of glucose phosphorylation in vitro. Aerobic growth of even the latter strain was largely fermentative (ca. 1 mol ethanol per mol glucose). Invertase expression was determined for a variety of media. All strains with HXK2 showed repression in growth on glucose and the others did not. Derepression in the wild-type strain occurred at ca. 1 mM glucose. The metabolic data do not support- or disprove-a model with HXK2 having only a secondary role in catabolite repression related to more rapid metabolism.
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Rizzetto, Lisa, Elena Zanni, Daniela Uccelletti, Ileana Ferrero y Paola Goffrini. "Extension of Chronological Lifespan by Hexokinase Mutation inKluyveromyces lactisInvolves Increased Level of the Mitochondrial Chaperonin Hsp60". Journal of Aging Research 2012 (2012): 1–10. http://dx.doi.org/10.1155/2012/946586.
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Oxidative damage, mitochondrial dysfunction, genomic instability, and telomere shortening represent all molecular processes proposed as causal factors in aging. Lifespan can be increased by metabolism through an influence on such processes. Glucose reduction extends chronological lifespan (CLS) ofSaccharomyces cerevisiaethrough metabolic adaptation to respiration. To answer the question if the reduced CLS could be ascribed to glucoseper seor to glucose repression of respiratory enzymes, we used theKluyveromyces lactisyeast, where glucose repression does not affect the respiratory function. We identified the unique hexokinase, encoded byRAG5gene, as an important player in influencing yeast lifespan by modulating mitochondrial functionality and the level of the mitochondrial chaperonin Hsp60. In this context, this hexokinase might have a regulatory role in the influence of CLS, shedding new light on the complex regulation played by hexokinases.
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Katz, Margaret E., Amir Masoumi, Stephen R. Burrows, Carolyn G. Shirtliff y Brian F. Cheetham. "The Aspergillus nidulans xprF Gene Encodes a Hexokinase-like Protein Involved in the Regulation of Extracellular Proteases". Genetics 156, n.º 4 (1 de diciembre de 2000): 1559–71. http://dx.doi.org/10.1093/genetics/156.4.1559.
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Abstract The extracellular proteases of Aspergillus nidulans are produced in response to limitation of carbon, nitrogen, or sulfur, even in the absence of exogenous protein. Mutations in the A. nidulans xprF and xprG genes have been shown to result in elevated levels of extracellular protease in response to carbon limitation. The xprF gene was isolated and sequence analysis indicates that it encodes a 615-amino-acid protein, which represents a new type of fungal hexokinase or hexokinase-like protein. In addition to their catalytic role, hexokinases are thought to be involved in triggering carbon catabolite repression. Sequence analysis of the xprF1 and xprF2 alleles showed that both alleles contain nonsense mutations. No loss of glucose or fructose phosphorylating activity was detected in xprF1 or xprF2 mutants. There are two possible explanations for this observation: (1) the xprF gene may encode a minor hexokinase or (2) the xprF gene may encode a protein with no hexose phosphorylating activity. Genetic evidence suggests that the xprF and xprG genes are involved in the same regulatory pathway. Support for this hypothesis was provided by the identification of a new class of xprG- mutation that suppresses the xprF1 mutation and results in a protease-deficient phenotype.
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Cárdenas, M. L. "Kinetic behaviour of vertebrate hexokinases with emphasis on hexokinase D (IV)". Biochemical Society Transactions 25, n.º 1 (1 de febrero de 1997): 131–35. http://dx.doi.org/10.1042/bst0250131.
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Marcus, Frank y Tito Ureta. "Amino acid sequence homology between yeast hexokinases and rat hexokinase C". Biochemical and Biophysical Research Communications 139, n.º 2 (septiembre de 1986): 714–19. http://dx.doi.org/10.1016/s0006-291x(86)80049-3.
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Lynch, R. M., W. Carrington, K. E. Fogarty y F. S. Fay. "Metabolic modulation of hexokinase association with mitochondria in living smooth muscle cells". American Journal of Physiology-Cell Physiology 270, n.º 2 (1 de febrero de 1996): C488—C499. http://dx.doi.org/10.1152/ajpcell.1996.270.2.c488.
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Hexokinase isoform I binds to mitochondria of many cell types. It has been hypothesized that this association is regulated by changes in the concentrations of specific cellular metabolites. To study the distribution of hexokinase in living cells, fluorophore-labeled functional hexokinase I was prepared. After microinjection into A7r5 smooth muscle cells, hexokinase localized to distinct structures identified as mitochondria. The endogenous hexokinase demonstrated a similar distribution with the use of immunocytochemistry. 2-Deoxyglucose elicited an increase in glucose 6-phosphate (G-6-P) and a decrease in ATP levels and diminished hexokinase binding to mitochondria in single cells. 3-O-methylglucose elicited slowly developing decreases in all three parameters. In contrast, cyanide elicited a rapid decrease in both ATP and hexokinase binding. Analyses of changes in metabolite levels and hexokinase binding indicate a positive correlation between binding and cell energy state as monitored by ATP. On the other hand, only in the presence of 2-deoxyglucose was the predicted inverse correlation between binding and G-6-P observed. Unlike the relatively large changes in distribution observed with the fluorescent-injected hexokinase, cyanide caused only a small decrease in the localization of endogenous hexokinase with mitochondria. These findings suggest that changes in the concentrations of specific metabolites can alter the binding of hexokinase I to specific sites on mitochondria. Moreover, the apparent difference in sensitivity of injected and endogenous hexokinase to changes in metabolites may reflect the presence of at least two classes of binding mechanisms for hexokinase, with differential sensitivity to metabolites.
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Ma, H., L. M. Bloom, C. T. Walsh y D. Botstein. "The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae". Molecular and Cellular Biology 9, n.º 12 (diciembre de 1989): 5643–49. http://dx.doi.org/10.1128/mcb.9.12.5643-5649.1989.
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Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.
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Ma, H., L. M. Bloom, C. T. Walsh y D. Botstein. "The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae." Molecular and Cellular Biology 9, n.º 12 (diciembre de 1989): 5643–49. http://dx.doi.org/10.1128/mcb.9.12.5643.
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Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.
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Liu, Chunyan, Xiuli Wang y Youzhong Zhang. "The Roles of HK2 on Tumorigenesis of Cervical Cancer". Technology in Cancer Research & Treatment 18 (1 de enero de 2019): 153303381987130. http://dx.doi.org/10.1177/1533033819871306.
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Cancer cells undergo metabolic changes that support their malignant growth. These changes are often associated with increased expression of the rate-limiting glycolytic enzyme hexokinase 2. Hexokinase 2 is an enzyme that catalyzes the conversion of glucose to glucose-6-phosphate. In this study, we utilized Gene Expression Profiling Interactive Analysis (GEPIA) database analysis and clinical sample analysis to find that hexokinase 2 was highly expressed in cervical cancer. Furthermore, we found that high hexokinase 2 expression in cervical cancer demonstrated a positive correlation with tumor size ( P = .009696), pathological grade ( P = .028551), and prognosis ( P = .00069) but not with age ( P = .956201) or lymph node metastasis ( P = .131379). At the cellular level, we knocked down the expression of hexokinase 2 in the human cervical cancer cell line SiHa. The results demonstrated that knockdown of hexokinase 2 inhibited the proliferation and migration of SiHa cells and promoted cell apoptosis. During this process, knockdown of hexokinase 2 inhibited phosphorylation of AKT and mammalian target of rapamycin and promoted p53 expression. At the same time, overexpression of human papillomavirus 18 oncogenes E6 and E7 significantly promoted the expression of hexokinase 2. Most importantly, we discovered a novel upstream regulatory microRNA for hexokinase 2: miR-9-5p. Luciferase reporter assays and Western blot assays demonstrated that hexokinase 2 expression was inhibited by miR-9-5p by directly binding its 3′-untranslated region in SiHa cells. Next, we determined that miR-9-5p could suppress the proliferation and migration of SiHa cells and induce apoptosis. In conclusion, we found that hexokinase 2 serves a carcinogenic role in cervical cancer through the miR-9-5p/hexokinase 2/AKT pathway, which serves as the basis for potential therapeutic targets and prognostic indicators.
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Pratidina, Ellen Ayuningtyas, Eko Suhartono y Bambang Setiawan. "IMPACT OF HEAVY METALS ON HEXOKINASE ISOFORMS: AN IN SILICO STUDY". Berkala Kedokteran 18, n.º 1 (21 de marzo de 2022): 29. http://dx.doi.org/10.20527/jbk.v18i1.12801.
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Abstract: Coal mining activities in South Kalimantan produce waste that is very dangerous if not processed wisely. Coal waste produces heavy metals cadmium and mercury that can pollute the environment. Heavy metals that enter the human body will cause negative impacts in the field of health such as the disruption of the glycolysis process in humans. The purpose of this study was determine the interaction of heavy metals which is cadmium and mercury against hexokinase enzymes using hexokinase enzymes type I, II, III with PDB ID : 4F9O, 2NZT, 3HM8 taken from Protein Data Bank and using the molecular docking website MIB: Metal Ion Binding Site Prediction and Docking server. Docking results will be visualized using chimera app version 1.15. Molecular docking of the heavy metals cadmium and mercury can interact with all three types of hexokinase enzymes. Cadmium metal ions bind hydrophobicly to amino acid residues of hexokinase enzymes type I, II, and III, while mercury metal ions bind covalently coordinate with amino acid residues of hexokinase enzymes type I and III. Mercury metal ions bind more strongly than cadmium metal ions. Of the three types of hexokinase enzymes, mercury metal ions bind most strongly with hexokinase enzyme type II because mercury ions bind to the active site of the three amino acid residues of hexokinase enzymes type II.Keywords: Cadmium ; hexokinase enzyme ; mercury ; molecular docking
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Lynch, R. M., K. E. Fogarty y F. S. Fay. "Modulation of hexokinase association with mitochondria analyzed with quantitative three-dimensional confocal microscopy." Journal of Cell Biology 112, n.º 3 (1 de febrero de 1991): 385–95. http://dx.doi.org/10.1083/jcb.112.3.385.
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Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.
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Kuettner, Bartholomeus, Karina Kettner, Thomas Kriegel y Norbert Sträter. "X-ray diffraction analysis of homodimeric yeast hexokinases". Acta Crystallographica Section A Foundations and Advances 70, a1 (5 de agosto de 2014): C410. http://dx.doi.org/10.1107/s2053273314095898.
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Yeast hexokinases are described in textbooks as classical open/close-switch monomeric enzymes. The hexokinase isoenzymes ScHxk1 and ScHxk2 of the budding yeast Saccharomyces cerevisiae share 77 % sequence identity. They exist in either an open or closed conformation, however, the published states were not of the same isoform. Crystal structures of the hexokinase KlHxk1 of the milk yeast Kluyvermyces lactis demonstrated open/close states of the same yeast hexokinase isoenzyme for the first time. KlHxk1 has 70 or 73 % sequence identity with ScHxk1 or 2, respectively. ScHxk2 and KlHxk1 contain an N-terminal binding stretch for the transcriptional repressor Mig1 which is the structural link to their function in glucose repression. They also exhibit monomer-dimer equilibria depending on protein concentration and phosphorylation of an N-terminal serine residue (S15). Experimental evidence for an expected ring-shaped homodimer of ScHxk2 was still missing to explain the phosphorylation-dependent oligomerization as demonstrated for KlHxk1 (figure). Structural data of other yeast glucose kinases were also limited in order to support a similar phenomenon. Therefore, we comparatively explored the oligomeric structure of ScHxk2 and K. lactis glucokinase 1 (KlGlk1) by means of X-ray diffraction in solution and in the crystalline state. The crystal structure of ScHxk2 was solved at high resolution (<1.5Å), and small-angle X-ray data of ScHxk2 were collected. Data analysis indicated that all three glucose-phosphorylating enzymes share a similar oligomeric architecture.
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MICCOLI, Laurent, Stéphane OUDARD, Franck SUREAU, Florence POIRSON, Bernard DUTRILLAUX y Marie-France POUPON. "Intracellular pH governs the subcellular distribution of hexokinase in a glioma cell line". Biochemical Journal 313, n.º 3 (1 de febrero de 1996): 957–62. http://dx.doi.org/10.1042/bj3130957.
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Hexokinase plays a key role in regulating cell energy metabolism. Hexokinase is mainly particulate, bound to the mitochondrial outer membrane in brain and tumour cells. We hypothesized that the intracellular pH (pHi) controls the intracellular distribution of hexokinase. Using the SNB-19 glioma cell line, pHi variations were imposed by incubating cells in a high-K+ medium at different pH values containing specific ionophores (nigericin and valinomycin), without affecting cell viability. Subcellular fractions of cell homogenates were analysed for hexokinase activity. Imposed pHi changes were verified microspectrofluorimetrically by using the pHi-sensitive probe SNARF-1-AM (seminaphthorhodafluor-1-acetoxymethyl ester). Imposition of an acidic pHi for 30 min strongly decreased the particulate/total hexokinase ratio, from 63% in the control sample to 31%. Conversely, when a basic pHi was imposed, the particulate/total hexokinase ratio increased to 80%. The glycolytic parameters, namely lactate/pyruvate ratio, glucose 6-phosphate and ATP levels, were measured concomitantly. Lactate/pyruvate ratio and ATP level were both markedly decreased by acidic pHi and increased by basic pHi. Conversely, the glucose 6-phosphate level was increased by acidic pHi and decreased by basic pHi. To demonstrate that the change of hexokinase distribution was not due to altered metabolite levels of glycolysis, a pHi was imposed for a 5 min incubation time. Modification of the hexokinase distribution was similar to that noted after a 30 min incubation, whereas metabolite levels of glycolysis were not affected. These results provide evidence that the intracellular distribution of hexokinase is highly sensitive to variations of the pHi, and regulates hexokinase activity.
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Magnani, M., V. Stocchi, L. Cucchiarini, G. Novelli, S. Lodi, L. Isa y G. Fornaini. "Hereditary nonspherocytic hemolytic anemia due to a new hexokinase variant with reduced stability". Blood 66, n.º 3 (1 de septiembre de 1985): 690–97. http://dx.doi.org/10.1182/blood.v66.3.690.690.
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Abstract A 27-year-old woman with severe chronic hemolytic anemia was found to have reduced red cell hexokinase activity when the degree of reticulocytosis was considered. This enzyme had normal pH-dependent activity, normal Km for glucose, fructose, and mannose, normal Km for Mg adenosine triphosphate (ATP)2- and Ki for glucose-1,6-diphosphate. Furthermore, the pH-dependence and orthophosphate dependence of Ki for glucose-1,6-diphosphate were normal. However, this hexokinase was inactivated rapidly at 44 degrees C. No abnormalities were found in the red cell hexokinase isozymic pattern when it was compared with the profile obtained from cells of similar age. The hexokinase specific activity was reduced in all the red blood cell fractions obtained by density gradient ultracentrifugation; a marked difference in the distribution of cells through the gradient was evident. Among the glycolytic intermediates, a significant decrease of 2,3- diphosphoglycerate was evident. ATP and glucose 6-phosphate were also reduced when compared with cells of similar. Glucose consumption of the hexokinase-deficient cells decreased, but the rate of glucose metabolized through the hexose monophosphate shunt was unchanged. Although the total hexokinase activity in lymphocytes was only reduced by 37%, a marked hexokinase deficiency was detected in blood platelets (20% to 25% of normal activity). The parents and one of two siblings of the patient were heterozygous for the defect, with 66% to 74% of normal erythrocyte hexokinase activity and reduced heat stability of the enzyme. These results, when compared with those obtained in previously reported cases of hexokinase deficiency, provide further evidence of the broad phenotypic variability that characterizes this disorder. Furthermore, it is suggested that failure of energy generation is probably the primary cause of hemolytic anemia in hexokinase deficiency.
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Magnani, M., V. Stocchi, L. Cucchiarini, G. Novelli, S. Lodi, L. Isa y G. Fornaini. "Hereditary nonspherocytic hemolytic anemia due to a new hexokinase variant with reduced stability". Blood 66, n.º 3 (1 de septiembre de 1985): 690–97. http://dx.doi.org/10.1182/blood.v66.3.690.bloodjournal663690.
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A 27-year-old woman with severe chronic hemolytic anemia was found to have reduced red cell hexokinase activity when the degree of reticulocytosis was considered. This enzyme had normal pH-dependent activity, normal Km for glucose, fructose, and mannose, normal Km for Mg adenosine triphosphate (ATP)2- and Ki for glucose-1,6-diphosphate. Furthermore, the pH-dependence and orthophosphate dependence of Ki for glucose-1,6-diphosphate were normal. However, this hexokinase was inactivated rapidly at 44 degrees C. No abnormalities were found in the red cell hexokinase isozymic pattern when it was compared with the profile obtained from cells of similar age. The hexokinase specific activity was reduced in all the red blood cell fractions obtained by density gradient ultracentrifugation; a marked difference in the distribution of cells through the gradient was evident. Among the glycolytic intermediates, a significant decrease of 2,3- diphosphoglycerate was evident. ATP and glucose 6-phosphate were also reduced when compared with cells of similar. Glucose consumption of the hexokinase-deficient cells decreased, but the rate of glucose metabolized through the hexose monophosphate shunt was unchanged. Although the total hexokinase activity in lymphocytes was only reduced by 37%, a marked hexokinase deficiency was detected in blood platelets (20% to 25% of normal activity). The parents and one of two siblings of the patient were heterozygous for the defect, with 66% to 74% of normal erythrocyte hexokinase activity and reduced heat stability of the enzyme. These results, when compared with those obtained in previously reported cases of hexokinase deficiency, provide further evidence of the broad phenotypic variability that characterizes this disorder. Furthermore, it is suggested that failure of energy generation is probably the primary cause of hemolytic anemia in hexokinase deficiency.
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Puri, R. N. y R. Roskoski. "Inactivation of yeast hexokinase by 2-aminothiophenol. Evidence for a ‘half-of-the-sites’ mechanism". Biochemical Journal 254, n.º 3 (15 de septiembre de 1988): 819–27. http://dx.doi.org/10.1042/bj2540819.
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Yeast hexokinase is a homodimer consisting of two identical subunits. Yeast hexokinase was inactivated by 2-aminothiophenol at 25 degrees C (pH 9.1). The reaction followed pseudo-first-order kinetics until about 70% of the phosphotransferase activity was lost. About 0.65 mol of 2-aminothiophenol/mol of hexokinase was found to be bound after the 70% loss of the enzyme activity. Completely inactivated hexokinase showed a stoichiometry of about 1 mol of 2-aminothiophenol bound/mol of the enzyme. The evidence obtained from kinetic experiments, stoichiometry of the inactivation reaction and fluorescence emission measurements suggested site-site interaction (weak negative co-operativity) during the inactivation reaction. The approximate rate constants for the reversible binding of 2-aminothiophenol to the first subunit (KI) and for the rate of covalent bond formation with only one site occupied (k3) were 150 microM and 0.046 min-1 respectively. The inactivation reaction was pH-dependent. Dithiothreitol, 2-mercaptoethanol and cysteine restored the phosphotransferase activity of the hexokinase after inactivation by 2-aminothiophenol. Sugar substrates protected the enzyme from inactivation more than did the nucleotides. Thus it is concluded that the inactivation of the hexokinase by 2-aminothiophenol was a consequence of a covalent disulphide bond formation between the aminothiol and thiol function at or near the active site of the enzyme. Hexokinase that had been completely inactivated by 2-aminothiophenol reacted with o-phthalaldehyde. Fluorescence emission intensity of the incubation mixture containing 2-aminothiophenol-modified hexokinase and o-phthalaldehyde was one-half of that obtained from an incubation mixture containing hexokinase and o-phthalaldehyde under similar experimental conditions. The intensity and position of the fluorescence emission maximum of the 2-aminothiophenol-modified hexokinase were different from those of the native enzyme, indicating conformational change following modification. Whereas aliphatic aminothiols were completely ineffective, aromatic aminothiols were good inhibitors of the hexokinase. Cyclohexyl mercaptan weakly inhibited the enzyme. Inhibition of the hexokinase by heteroaromatic thiols was dependent on the nature of the heterocyclic ring and position of the thiol-thione equilibrium. The inhibitory function of a thiol is associated with the following structural characteristics: (a) the presence of an aromatic ring, (b) the presence of a free thiol function and (c) the presence of a free amino function in the close proximity of the thiol function.(ABSTRACT TRUNCATED AT 400 WORDS)
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Rempel, A., P. Bannasch y D. Mayer. "Microheterogeneity of cytosolic and membrane-bound hexokinase II in Morris hepatoma 3924A". Biochemical Journal 303, n.º 1 (1 de octubre de 1994): 269–74. http://dx.doi.org/10.1042/bj3030269.
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Phosphorylation of glucose by hexokinase is the key step in glucose and energy metabolism of the cell. In the Morris hepatoma 3924A, hexokinase II is the predominant hexokinase isoenzyme and occurs in the cytosol as well as bound to membranes. Hexokinase II was isolated by DEAE-cellulose chromatography from both the cytosolic and the mitochondria-enriched fractions and further resolved by hydrophobic-interaction chromatography on phenyl-Sepharose into two components designated hexokinase IIa and IIb. In both the soluble and the mitochondria-enriched fractions, type IIb was the predominant form, but the IIb/IIa ratio was higher in the particulate (6-8) as compared with the cytosolic fraction (1.5-2.0). Binding of the isolated forms of the enzyme to rat liver mitochondria resulted in a 2-10-fold activation of both subtypes. Biochemical characterization showed that both subtypes are closely related to the isoenzyme commonly referred to as hexokinase II, and that the microheterogeneity was not a consequence of contamination with hexokinase I or III. Both subtypes had a molecular mass of 110 kDa, they were inhibited by Pi at concentrations higher than 5 mM, and activated by the detergent CHAPS. The two subtypes differed in electrophoretic mobility (IIa > IIb), in Km values for glucose (IIa, 0.109 mM; IIb, 0.216 mM), in Ki values for glucose 6-phosphate (IIa, 25 microM; IIb, 0.106 mM), and in Ki values for glucose 1,6-biphosphate (IIa, 12.2 microM; IIb, 5.5 microM). An artificial proteolytic cleavage as cause of the hexokinase II microheterogeneity can be excluded, since both subtypes show the same molecular mass and the ability to bind to mitochondria and phenyl-Sepharose. In addition, the relative proportions of the two subtypes did not vary markedly between several enzyme preparations. Northern-blot analysis with a hexokinase II-specific cDNA probe revealed two distinct mRNA transcripts of 5.2 and 6.3 kb in length, which offers the possibility that hexokinase II microheterogeneity is due to differential RNA transcription and/or processing.
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Entian, K. D., F. Hilberg, H. Opitz y D. Mecke. "Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression". Molecular and Cellular Biology 5, n.º 11 (noviembre de 1985): 3035–40. http://dx.doi.org/10.1128/mcb.5.11.3035-3040.1985.
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The regulatory hexokinase PII mutants isolated previously (K.-D. Entian and K.-U. Fröhlich, J. Bacteriol. 158:29-35, 1984) were characterized further. These mutants were defective in glucose repression. The mutation was thought to be in the hexokinase PII structural gene, but it did not affect the catalytic activity of the enzyme. Hence, a regulatory domain for glucose repression was postulated. For further understanding of this regulatory system, the mutationally altered hexokinase PII proteins were isolated from five mutants obtained independently and characterized by their catalytic constants and bisubstrate kinetics. None of these characteristics differed from those of the wild type, so the catalytic center of the mutant enzymes remained unchanged. The only noticeable difference observed was that the in vivo modified form of hexokinase PII, PIIM, which has been described recently (K.-D. Entian and E. Kopetzki, Eur. J. Biochem. 146:657-662, 1985), was absent from one of these mutants. It is possible that the PIIM modification is directly connected with the triggering of glucose repression. To establish with certainty that the mutation is located in the hexokinase PII structural gene, the genes of these mutants were isolated after transforming a hexokinaseless mutant strain and selecting for concomitant complementation of the nuclear function. Unlike hexokinase PII wild-type transformants, glucose repression was not restored in the hexokinase PII mutant transformants. In addition mating experiments with these transformants followed by tetrad analysis of sporulated diploids gave clear evidence of allelism to the hexokinase PII structural gene.
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Entian, K. D., F. Hilberg, H. Opitz y D. Mecke. "Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression." Molecular and Cellular Biology 5, n.º 11 (noviembre de 1985): 3035–40. http://dx.doi.org/10.1128/mcb.5.11.3035.
Texto completoResumen
The regulatory hexokinase PII mutants isolated previously (K.-D. Entian and K.-U. Fröhlich, J. Bacteriol. 158:29-35, 1984) were characterized further. These mutants were defective in glucose repression. The mutation was thought to be in the hexokinase PII structural gene, but it did not affect the catalytic activity of the enzyme. Hence, a regulatory domain for glucose repression was postulated. For further understanding of this regulatory system, the mutationally altered hexokinase PII proteins were isolated from five mutants obtained independently and characterized by their catalytic constants and bisubstrate kinetics. None of these characteristics differed from those of the wild type, so the catalytic center of the mutant enzymes remained unchanged. The only noticeable difference observed was that the in vivo modified form of hexokinase PII, PIIM, which has been described recently (K.-D. Entian and E. Kopetzki, Eur. J. Biochem. 146:657-662, 1985), was absent from one of these mutants. It is possible that the PIIM modification is directly connected with the triggering of glucose repression. To establish with certainty that the mutation is located in the hexokinase PII structural gene, the genes of these mutants were isolated after transforming a hexokinaseless mutant strain and selecting for concomitant complementation of the nuclear function. Unlike hexokinase PII wild-type transformants, glucose repression was not restored in the hexokinase PII mutant transformants. In addition mating experiments with these transformants followed by tetrad analysis of sporulated diploids gave clear evidence of allelism to the hexokinase PII structural gene.
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Chambers, Jeremy W., Margaret T. Kearns, Meredith T. Morris y James C. Morris. "Assembly of Heterohexameric Trypanosome Hexokinases Reveals That Hexokinase 2 Is a Regulable Enzyme". Journal of Biological Chemistry 283, n.º 22 (3 de abril de 2008): 14963–70. http://dx.doi.org/10.1074/jbc.m802124200.
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Morris, Meredith T., Courtney DeBruin, Zhaoqing Yang, Jeremy W. Chambers, Kerry S. Smith y James C. Morris. "Activity of a Second Trypanosoma brucei Hexokinase Is Controlled by an 18-Amino-Acid C-Terminal Tail". Eukaryotic Cell 5, n.º 12 (6 de octubre de 2006): 2014–23. http://dx.doi.org/10.1128/ec.00146-06.
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ABSTRACT Trypanosoma brucei expresses two hexokinases that are 98% identical, namely, TbHK1 and TbHK2. Homozygous null TbHK2−/− procyclic-form parasites exhibit an increased doubling time, a change in cell morphology, and, surprisingly, a twofold increase in cellular hexokinase activity. Recombinant TbHK1 enzymatic activity is similar to that of other hexokinases, with apparent Km values for glucose and ATP of 0.09 ± 0.02 mM and 0.28 ± 0.1 mM, respectively. The k cat value for TbHK1 is 2.9 × 104 min−1. TbHK1 can use mannose, fructose, 2-deoxyglucose, and glucosamine as substrates. In addition, TbHK1 is inhibited by fatty acids, with lauric, myristic, and palmitic acids being the most potent (with 50% inhibitory concentrations of 75.8, 78.4, and 62.4 μM, respectively). In contrast to TbHK1, recombinant TbHK2 lacks detectable enzymatic activity. Seven of the 10 amino acid differences between TbHK1 and TbHK2 lie within the C-terminal 18 amino acids of the polypeptides. Modeling of the proteins maps the C-terminal tails near the interdomain cleft of the enzyme that participates in the conformational change of the enzyme upon substrate binding. Replacing the last 18 amino acids of TbHK2 with the corresponding residues of TbHK1 yields an active recombinant protein with kinetic properties similar to those of TbHK1. Conversely, replacing the C-terminal tail of TbHK1 with the TbHK2 tail inactivates the enzyme. These findings suggest that the C-terminal tail of TbHK1 is important for hexokinase activity. The altered C-terminal tail of TbHK2, along with the phenotype of the knockout parasites, suggests a distinct function for the protein.
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Puri, R. N. y R. Roskoski. "Inactivation of yeast hexokinase by Cibacron Blue 3G-A: spectral, kinetic and structural investigations". Biochemical Journal 300, n.º 1 (15 de mayo de 1994): 91–97. http://dx.doi.org/10.1042/bj3000091.
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Yeast hexokinase, a homodimer (100 kDa), is an important enzyme in the glycolytic pathway. Although Cibacron Blue 3G-A (Reactive Blue 2) has been previously shown to inactivate yeast hexokinase, no comprehensive study exists concerning the nature of interaction(s) between hexokinase and the blue dye. A comparison of the computer-generated three-dimensional (3D) representations showed considerable overlap of the purine ring of ATP, a nucleotide substrate of hexokinase, with the hydrophobic anthraquinone moiety of the blue dye. The visible spectrum of the blue dye showed a characteristic absorption band centred at 628 nm. The visible difference spectrum of increasing concentration of the dye and the same concentrations of the dye plus a fixed concentration of hexokinase exhibited a maximum, a minimum and an isobestic point at 683, 585, and 655 nm respectively. The visible difference spectrum of the blue dye and the dye in 50% ethylene glycol showed a maximum and a minimum at 660 and 570 nm respectively. The visible difference spectrum of the blue dye in the presence of the dye and hexokinase modified at the active site by pyridoxal phosphate, iodoacetamide and o-phthalaldehyde was devoid of bands characteristic of the hexokinase-blue dye complex. Size-exclusion-chromatographic studies in the absence or presence of guanidinium chloride showed that the enzyme inactivated by the blue dye was co-eluted with the unmodified enzyme. The dialysis residue obtained after extensive dialysis of the gel-filtered complex, against a buffer of high ionic strength, showed an absorption maximum at 655 nm characteristic of the dye-enzyme complex. Inactivation data when analysed by ‘Kitz-Wilson’-type kinetics for an irreversible inhibitor, yielded values of 0.05 min-1 and 92 microM for maximum rate of inactivation (k3) and dissociation constant (Kd) for the enzyme-dye complex respectively. Sugar and nucleotide substrates protected hexokinase against inactivation by the blue dye. About 2 mol of the blue dye bound per mol of hexokinase after complete inactivation. The inactivated enzyme could not be re-activated in the presence of 1 M NaCl. These results suggest that Cibacron Blue 3G-A inactivated hexokinase by an irreversible adduct formation at or near the active-site. Spectral and kinetic studies coupled with an analysis of the 3D representations of model compounds corresponding to the substructures of the blue dye suggest that 1-amino-4-(N-phenylamino)anthraquinone-2-sulphonic acid part of the blue dye may represent the minimum structure of Cibacron Blue 3G-A necessary to bind hexokinase.(ABSTRACT TRUNCATED AT 400 WORDS)
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Lema, M. García de, G. Lucchesi, G. Racagni y E. E. Machado-Domenech. "Changes in enzymatic activities involved in glucose metabolism by acyl-CoAs in Trypanosoma cruzi". Canadian Journal of Microbiology 47, n.º 1 (1 de enero de 2001): 49–54. http://dx.doi.org/10.1139/w00-120.
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This study describes the effect of some saturated and unsaturated free fatty acids and acyl-CoA thioesters on Trypanosoma cruzi glucose 6-phosphate dehydrogenase and hexokinase activities. Glucose 6-phosphate dehydrogenase was sensitive to the destabilizing effect provoked by free fatty acids, while hexokinase remained unaltered. Glucose 6-phosphate dehydrogenase inhibition by free fatty acids was dependent on acid concentration and chain length. Both enzymes were inhibited when they were incubated with acyl-CoA thioesters. The acyl-CoA thioesters inhibited glucose 6-phosphate dehydrogenase at a lower concentration than the free fatty acids; the ligands glucose 6-phosphate and NADP+ afforded protection. The inhibition of hexokinase by acyl-CoAs was not reverted when the enzyme was incubated with ATP. The type of inhibition found with acyl-CoAs in relation to glucose 6-phosphate dehydrogenase and hexokinase suggests that this type inhibition may produce an in vivo modulation of these enzymatic activities.Key words: Trypanosoma cruzi, fatty acids, acyl-CoAs, glucose 6-phosphate dehydrogenase, hexokinase.
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Youderian, Philip, Matthew C. Lawes, Chad Creighton, Jessica C. Cook y Milton H. Saier. "Mutations That Confer Resistance to 2-Deoxyglucose Reduce the Specific Activity of Hexokinase from Myxococcus xanthus". Journal of Bacteriology 181, n.º 7 (1 de abril de 1999): 2225–35. http://dx.doi.org/10.1128/jb.181.7.2225-2235.1999.
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ABSTRACT The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of Myxococcus xanthus. Mutants ofM. xanthus resistant to 2dGlc, designated hexmutants, arise at a low spontaneous frequency. Expression of theEscherichia coli glk (glucokinase) gene in M. xanthus hex mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of M. xanthusextracts reveal a soluble hexokinase (ATP:d-hexose-6-phosphotransferase; EC 2.7.1.1 ) activity but no phosphotransferase system activities. The hexmutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the hexmutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc andN-acetylglucosamine act as inhibitors of glucose turnover by the M. xanthus hexokinase in vitro, consistent with the finding that glucose and N-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo.
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Arora, Krishan K., Glenn L. Decker y Peter L. Pedersen. "Glucose metabolism in cancer cells: immunogold localization of hexokinase to the outer mitochondrial membrane". Proceedings, annual meeting, Electron Microscopy Society of America 53 (13 de agosto de 1995): 1044–45. http://dx.doi.org/10.1017/s0424820100141597.
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Hexokinase (ATP: D-hexose 6-phophotransferase EC 2.7.1.1) is the first enzyme of the glycolytic pathway which commits glucose to catabolism by catalyzing the phosphorylation of glucose with ATP. Previous studies have shown diat hexokinase activity is markedly elevated in rapidly growing tumor cells exhibiting high glucose catabolic rates. A large fraction (50-80%) of this enzyme activity is bound to the mitochondrial fraction (1,2) where it has preferred access to ATP (3). In contrast,the hexokinase activity of normal tissues is quite low, with one exception being brain which is a glucose-utilizing tissue (4). Biochemical evidence involving rigorous subfractionation studies have revealed striking differences between the subcellular distribution of hexokinase in normal and tumor cells [See review by Arora et al (4)].In the present report, we have utilized immunogold labeling techniques to evaluate die subcellular localization of hexokinase in highly glycolytic AS-30D hepatoma cells and in the tissue of its origin, i.e., rat liver.
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Patra, Krushna C. y Nissim Hay. "Hexokinase 2 as oncotarget". Oncotarget 4, n.º 11 (31 de octubre de 2013): 1862–63. http://dx.doi.org/10.18632/oncotarget.1563.
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van Veelen, C. W., G. Rijksen, E. Sprengers y G. E. Staal. "Hexokinase in cultured gliomas". Neurosurgery 18, n.º 3 (marzo de 1986): 389. http://dx.doi.org/10.1097/00006123-198603000-00034.
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