Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.1.1 (hexokinase)
 5,274 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mature erythrocyte of the pig has been observed to possess the slowest metabolic rate of any mammalian cell type. Previous studies in this laboratory suggested that the hexokinase isolated from these cells was inhibited by glucose in concentrations in excess of 0.2 mM. In the present study, the enzyme was isolated by utilizing DEAE-Sephadex A-50, ammonium sulfate precipitation, DEAE-cellulose (DE-52), and Sephadex G-100 gel-filtration. Studies on the hexokinase isolated from the pig mature erythrocyte by the above procedures revealed two distinct isozymes of hexokinase that do not behave kinetically and electrophoretically as those previously found in other mammalian red blood cells. The isozyme isolated from the erythrocyte of the young adult pig (less than six months of age) migrated at a slower electrophoretic rate than the one isolated from the adult pig (more than six months of age). Coupled with the observed difference in electrophoretic mobilities were changes in the apparent Km values as well as Vmax as a function of substrate concentration. In spite of the changes observed in relation to glucose, the apparent Km for Mg-ATP-2 was not altered during development. Diphosphoglycerate (DPG) was observed to be a "linear-mixed" inhibitor of both isozymes with respect to Mg-ATP-2. An experimental designed to determined the type of inhibition by DPG on the type I isozyme isolated from the horse erythrocyte revealed competitive inhibition with the Mg-ATP-2 site. Free Mg activated both isozymes in low concentrations (less than 2.5 mM) but inhibited the enzymatic activity as the concentration was elevated. The data suggest that both the young adult and the adult pig erythrocyte possess two distinct type III isozymes of hexokinase.
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PMID:Erythrocyte metabolism: kinetic and electrophoretic analyses of pig red cell hexokinase. 697 14

Rat hexokinases fro caput sperm (immature) and caudal sperm (mature) were investigated. The hexokinases from both sources were studied by DEAE-cellulose column chromatography and by cellulose acetate electrophoresis. The specific activity of caput sperm hexokinase was not significantly different from that of the caudal sperm enzyme. Spermatozoa possess two isozymes of hexokinase. Type I hexokinase was the predominant type in caput sperm whereas the sperm type of hexokinase was predominant in caudal sperm. Hexokinase types II and III were absent in both extracts.
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PMID:Increase in sperm type hexokinase activity of rat spermatozoa during maturation. 720 84

Two major hexokinases (ATP: D-hexose 6-phosphotransferases, EC 2.7.1.1) have been identified in tissues of Homarus americanus (lobster) and separated from each other by DEAE-cellulose ion-exchange chromatography and by polyacrylamide gel electrophoresis. The molecular weight of each, determined by gel filtration, is about 50 000. Hexokinase II, named for its column elution order, resembles hexokinase isozymes I and II of vertebrates. Km values for glucose, mannose and fructose are 0.08, 0.13 and 6.7 mM, respectively. It is strongly inhibited by the reaction products, ADP and glucose-6-P (Ki = 0.8 mM). Hexokinase I appears to be different from any animal hexokinase previously described. It has a high affinity for mannose and fructose and low affinity for glucose. Km values are 6, 0.07 and 1.2 mM and relative maximum rates 100, 520 and 1070 for glucose, mannose and fructose, respectively. Hexokinase I is not inhibited by physiological concentrations of ATP nor by glucose-6-P , mannose-6-P or fructose-6-P even at high concentrations. Both enzymes occur in muscle at about 10% of the concentration found in the hepatopancreas. The use of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49), with NAD as cofactor, is recommended for measuring hexokinases in crude tissue preparations to avoid the variable further reduction of nucleotide caused by the action of 6-phosphogluconate dehydrogenase when NADP is used with yeast glucose-6-phosphate dehydrogenase.
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PMID:Two hexokinases of Homarus americanus (lobster), one having great affinity for mannose and fructose and low affinity for glucose. 721 58

Glucose phosphorylating activities were measured in liver extracts from chicks at several developmental stages. Enzyme activity levels in supernates were low (about 0.16 units/g liver) from day 10th of egg incubation until the 17th day, at which time a transient increase to 0.5 units/g was observed. At hatching, the levels were again low (0.15 units/g) compared to adult levels (0.9 units/g). Particulate hexokinase activity was rather constant from day 10th to adulthood (about 0.3 units/g). Chromatography of liver supernates in DEAE-cellulose columns revealed the presence of four hexokinases in embryos up to day 15th of incubation. From that day onwards, the least retained from (hexokinase 4) was no longer found. The most retained form (hexokinase 1) disappeared at hatching, at which time a pattern consisting of hexokinases 2 and 3 was found to be very similar to the adult profile. The four isozymes were characterized as low Km glucose hexokinase of broad sugar specificities and molecular weights of about 100,000. Particulate hexokinase activity of embryonic chick liver was found to be composed of the same isozymes observed in cytosolic extracts. Incubation of particles with glucose 6-P or ATP failed to release hexokinase activity.
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PMID:Ontogeny of chick liver hexokinase isozymes. 734 73

The distribution of hexokinase isoenzymes was analyzed in erythrocytes from adults and newborn infants. Isolation by DEAE-cellulose chromatography revealed one main peak (I a) in erythrocytes from adults similar to hexokinase isoenzyme I from rat liver and a smaller peak (I b) which was eluted at a lower conductivity than rat liver isoenzyme II. In erythrocytes from newborn infants the peak I b dominated, isoenzyme I a was the smaller one. In adult erythrocytes an additional isoenzyme was detected which resembled rat liver isoenzyme III, this isoenzyme was absent in erythrocytes from newborn infants. The erythrocytic isoenzymes I a and I b differed in their kinetic properties. Isoenzyme I a had a slightly higher affinity for glucose (Km 47 microM) than isoenzyme I b (Km 100 microM). In the presence of 5mM Pi isoenzyme I b was more inhibited by glucose 6-phosphate than isoenzyme I a. 2.3-Diphosphoglycerate inhibited isoenzyme I b more than isoenzyme I a.
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PMID:Different biochemical properties of foetal and adult red cell hexokinase isoenzymes. 739 4

The liver of rainbow trout contains two hexokinases (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) designated C and D from the elution pattern in DEAE-cellulose column chromatography. Hexokinase D has been purified about 50-fold from the liver of rainbow trout by chromatography with DEAE-cellulose and Sephadex G-200, and by isoelectric focusing. The properties of hexokinase D were similar to those of mammalian hexokinase III with respect to the Km values for ATP and glucose and the substrate inhibition by glucose at high concentration. However, the enzyme showed a wide specificity for nucleotides as the phosphoryl donor. Although it has been reported that the only effective nucleotide as the phosphoryl donor for hexokinase from various origin in ATP, and that ADP, a reaction product, inhibits the enzyme, hexokinase D from the rainbow-trout liver was found to be able to form glucose 6-phosphate (Glc-6-P) from glucose and various nucleotides such as ATP, ADP, CTP, GTP, UTP and UDP. The reaction products from ADP and glucose, Glc-6-P and AMP, were identified by chromatography on ion-exchange resin column and paper. The enzyme D was not inhibited by ADP but was strongly inhibited by AMP, which is a reaction product from ADP.
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PMID:A hexokinase from fish liver with wide specificity for nucleotides as phosphoryl donor. 742 68

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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PMID:Microheterogeneity of cytosolic and membrane-bound hexokinase II in Morris hepatoma 3924A. 794 51

In vitro incubation of isolated hexokinase isozyme I or isolated dimer of mitochondrial creatine kinase with the outer mitochondrial membrane pore led to high molecular weight complexes of enzyme oligomers. Similar complexes of hexokinase and mitochondrial creatine kinase could be extracted by 0.5% Triton X-100 from homogenates of rat brain. Hexokinase and creatine kinase complexes could be separated by subsequent chromatography on DEAE anion exchanger. The molecular weight, as determined by gel-permeation chromatography, was approximately 400 kDa for both complexes. The Mr suggested tetramers of hexokinase (monomer 100 kDa) and creatine kinase (active enzyme is a dimer of 80 kDa). The composition of the complexes was further characterised by specific antibodies. Besides either hexokinase or creatine kinase molecules the complexes contained porin and adenylate translocator. It was possible to incorporate the complexes into artificial bilayer membranes and to measure conductance in 1 M KCI. The incorporating channels had a high conductance of 6 nS that was asymmetrically voltage dependent. The complexes were also reconstituted in phospholipid vesicles that were loaded with ATP. Complex containing vesicles retained ATP while vesicles reconstituted with pure porin were leaky. The internal ATP could be used by creatine kinase and hexokinase in the complex to phosphorylate external creatine or glucose. This process was inhibited by atractyloside. The hexokinase complex containing vesicles were furthermore loaded with malate or ATP that was gradually released by addition of Ca2+ between 100 and 600 microM. The liberation of malate or ATP by Ca2+ could be inhibited by N-methylVal-4-cyclosporin, suggesting that the porin translocator complex constitutes the permeability transition pore. The results show the physiological existence of kinase porin translocator complexes at the mitochondrial surface. It is assumed that such complexes between inner and outer membrane components are the molecular basis of contact sites observed by electron microscopy. Kinase complex formation may serve three regulatory functions, firstly regulation of the kinase activity, secondly stimulation of oxidative phosphorylation and thirdly regulation of the permeability transition pore.
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PMID:Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore. 891 85

The isozymes of three glycolytic regulatory kinases: hexokinase, phosphofructokinase and pyruvate kinase are fractionated by a single ion exchange chromatographic procedure on DEAE-cellulose. Enriched-erythroblast bone marrow cells showed two heterogeneous peaks, each consisting of two overlapping peaks: one major and one minor peak, but only two isozymes were observed in reticulocytes and erythrocytes. Phosphofructokinase showed multiple isozymic forms in the three cell populations, but while in erythroblasts the main one eluted in the last fractions, in reticulocytes and erythrocytes it eluted in the early fractions. Pyruvate kinase showed a main early activity peak with a shoulder in erythroblasts, reticulocytes and erythrocytes but the response to the allosteric effectors (fructose-1,6-bisphosphate and ATP) suggests the presence of different pyruvate kinase isozymes in reticulocytes and erythrocytes.
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PMID:A comparative study by a single chromatographic procedure of glycolytic regulatory kinase isozymes in rat erythroid cells as a function of differentiation-maturation process. 976 20

Hexokinase type I (HK I; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), the predominant glucose-phosphorylating enzyme in red blood cells, exists in human erythrocytes in multiple molecular forms that differ in isoelectric point and are separable by ion-exchange chromatography. The major forms, designated HK Ia, Ib and Ic, have similar kinetic properties but are characterized by different age-dependent decay and different intracellular distribution in reticulocytes. HK Ib, which elutes between HK I and HK II in the DEAE ion-exchange chromatography, appears to be unique to RBCs and different from any other hexokinase isozyme previously described. Indeed, Murakami and Piomelli recently reported the presence of a specific HK isozyme (named HKr) expressed in K562 cells and in human reticulocytes and, moreover, the resolution of the human HK I gene structure provided the direct evidence of an erythroid-specific exon 1. To further investigate the microheterogeneity of HK I in human RBCs we established a prokaryotic expression system for the HKr isozyme, using the pET plasmid, inducible with IPTG. The recombinant HKr, expressed in bacterial cells as a catalytically active enzyme, was purified to homogeneity by a combination of DEAE ionexchange chromatography followed by hydrophobic interaction chromatography and dye-ligand affinity chromatography. The kinetic and chromatographic properties of the homogeneous recombinant HKr suggest that this erythroid-specific HK isozyme in fact corresponds to the HK isoform previously described in human RBCs and referred to as HK Ib.
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PMID:Expression, purification, and characterization of a recombinant erythroid-specific hexokinase isozyme. 985 93


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