Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C.

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.     

Purification and physical properties of hexokinase from human erythrocytes

A bulk purification is described for hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from human erythrocytes. Following a 110,000-fold purification from 40 litres of blood, 5 mg protein with a specific activity of 22 units/mg were obtained. On application of various separation techniques, the enzyme activity co-migrated with the main protein component. The physical properties, such as the relative molecular mass of 108,000 and sedimentation coefficient of 5.5 S, are similar to those of the enzyme from human heart. In particular, there is a correspondence in the conformational response to glucose 6-phosphate as shown by an association of the enzyme promoted by this metabolite.     

Purification of skeletal muscle hexokinase by affinity elution chromatography

Type II hexokinase (EC 2.7.1.1) has been purified from rat skeletal muscle by a simple procedure involving chromatography on DEAE-cellulose, affinity elution chromatography from phosphocellulose, and gel filtration on Sephadex G-200. The key to the preparation of homogeneous enzyme is the affinity elution step in which an effector molecule, glucose 6-phosphate, is used as the eluting ligand. A 5300-fold purification is obtained by the procedure and over 400-fold purification is obtained in the affinity elution step alone. Approximately 3.3 mg of homogeneous hexokinase with a specific activity of 120 units/mg is obtained from 800 g of rat limb.     

Distribution of hexokinase isoenzymes depending on a carbon source in Saccharomyces cerevisiae.

DEAE cellulose chromatography and agar gel electrophoresis of glucose-phosphorylating enzymes in Saccharomyces cerevisiae showed the existence of glucokinase and two hexokinase isoenzymes ( designated as hexokinase I and II ). The distribution of hexokinase isoenzymes was dependent on a carbon source in the medium, while that of glucokinase was not dependent. The cells grown on 3 % ethanol as carbon source showed the isoenzyme pattern with predominant hexokinase I and a little hexokinase II. The isoenzyme pattern of the cells grown on 6 % glucose, which was differnt from that of the cells grown on ethanol, showed that hexokinase I and II were minor and major parts respectively. When the cells grown on 3 % ethanol were incubated on the medium containing 6 % glucose, hexokinase I was repressed and hexokinase II inducted. These facts suggest that two hexokinase isoenzymes, but not glucokinase, are adaptive enzyme.     

Determination of the Deoxyglucose and Glucose Phosphorylation Ratio and the Lumped Constant in Rat Brain and a Transplantable Rat Glioma

Mitochondrially bound hexokinase (ATP-D-hexose-6-phosphotransferase; EC 2.7.1.1) was dissociatively extracted from normal rat brains and intracerebral and subcutaneous implants of the 36B-10 glioma. At least 70% of the total hexokinase enzyme activity in normal and glioma tissue was associated with the mitochondrial fraction. Purification of the crude tissue extracts by ion-exchange and affinity chromatography followed by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a successive purification of the enzyme to homogeneity with a molecular size of 98 kilodaltons. Enzyme kinetics with glucose or 2-deoxyglucose (2-DG) as the substrate were measured spectrophotometrically by coupling the appropriate reactions to either NADPH or NAD+ formation. The Km of hexokinase with glucose as the substrate in the intracerebral glioma (0.138 mM) and subcutaneous glioma (0.183 mM) tissues was 2.1-2.7-fold higher than that observed in normal brain tissue (0.067 mM) (p less than 0.001). No significant differences were observed in the Km for hexokinase with 2-DG as the substrate in the glioma and normal brain tissue. The phosphorylation ratio for normal brain was 0.320 and was increased in the intracerebral glioma to 0.694 and in the subcutaneous glioma to 0.519. The ratios of deoxyglucose and glucose volumes of distribution in normal brain and intracerebral glioma tissues were 1.70 and 1.85, respectively. The lumped constants calculated directly from the phosphorylation ratios and the volumes of distribution of deoxyglucose and glucose were 0.517 in normal brain and 1.168 in intracerebral glioma. Our results indicate the lumped constant is increased 2.26-fold in intracerebral glioma compared with normal brain.     

Is hexokinase present in the basal lateral membranes of rat kidney proximal tubular epithelial cells?

The possible presence of hexokinase in basal lateral membranes from rat kidney proximal tubules was investigated. Basal lateral membranes were obtained from homogenates of rat kidney cortex by differential centrifugation and free flow electrophoresis. They were further purified by density gradient centrifugation. Hexokinase activity was measured as the phosphorylation of D-[U14C]glucose. Throughout the purification of the membranes, the specific activity of hexokinase decreased while that of (Na+ + K+)-ATPase increased. Hexokinase activity in all fractions could be quantitatively accounted for in terms of cytosolic and mitochondrial enzyme contributions. It is concluded that there is no hexokinase activity in basal lateral membranes from rat kidney.     

Nonaggregating mutant of recombinant human hexokinase I exhibits wild-type kinetics and rod-like conformations in solution.

Hexokinase I governs the rate-limiting step of glycolysis in brain tissue, being inhibited by its product, glucose 6-phosphate, and allosterically relieved of product inhibition by phosphate. On the basis of small-angle X-ray scattering, the wild-type enzyme is a monomer in the presence of glucose and phosphate at protein concentrations up to 10 mg/mL, but in the presence of glucose 6-phosphate, is a dimer down to protein concentrations as low as 1 mg/mL. A mutant form of hexokinase I, specifically engineered by directed mutation to block dimerization, remains monomeric at high protein concentration under all conditions of ligation. This nondimerizing mutant exhibits wild-type activity, potent inhibition by glucose 6-phosphate, and phosphate reversal of product inhibition. Small-angle X-ray scattering data from the mutant hexokinase I in the presence of glucose/phosphate, glucose/glucose 6-phosphate, and glucose/ADP/Mg2+/AlF3 are consistent with a rodlike conformation for the monomer similar to that observed in crystal structures of the hexokinase I dimer. Hence, any mechanism for allosteric regulation of hexokinase I should maintain a global conformation of the polypeptide similar to that observed in crystallographic structures.     

The overexpressed hexahistidine-tagged human hexokinase type III is inhibited by D-glucose

Inhibition by its product, glucose, is a kinetic property of hexokinase type III. In this paper, we report the overexpression in Escherichia coli of human hexokinase type III. The recombinant enzyme was genetically fused with a hexahistidine peptide at the C-terminal end. This modification confers to the product the ability to bind the Ni2+ ion immobilised into agarose by nitrilotriacetic acid (NTA) groups. The purification was performed by one-step column chromatography using ammonium sulphate as stabilising agent. Recombinant hexokinase type III appears as a single band of approximately 100 kDa on a SDS-PAGE gel and shows specific activity of 16 U/mg. Its kinetic parameters are comparable to those of the native enzyme, including the fact that it can be inhibited by glucose. The comparison of these results with the properties of the overexpressed carboxyl-domain led us to suppose that the inhibition site for glucose required the presence of the N-terminal domain.     

Factors affecting the glucose 6-phosphate inhibition of hexokinase from cerebral cortex tissue of the guinea pig

1. The inhibition of hexokinase by glucose 6-phosphate has been investigated in crude homogenates of guinea-pig cerebral cortex by using a sensitive radio-chemical technique for the assay of hexokinase activity. 2. It was observed that 44% of cerebral-cortex hexokinase activity did not sediment with the microsomal or mitochondrial fractions (particulate fraction), and this is termed soluble hexokinase. The sensitivities of soluble and particulate hexokinase, and hexokinase in crude homogenates, to the inhibitory actions of glucose 6-phosphate were measured; 50% inhibition was produced by 0.023, 0.046 and 0.068mm-glucose 6-phosphate for soluble, particulate and crude homogenates respectively. 3. The optimum Mg(2+) concentration for the enzyme was about 10mm, and this appeared to be independent of the ATP concentration. In the presence of added glucose 6-phosphate, raising the Mg(2+) concentration to 5mm increased the activity of hexokinase, but above this concentration Mg(2+) potentiated the glucose 6-phosphate inhibition. When present at a concentration above 1mm, Ca(2+) ions inhibited the enzyme in the presence or absence of glucose 6-phosphate. 4. When the ATP/Mg(2+) ratio was 1.0 or below, variations in the ATP concentration had no effect on the glucose 6-phosphate inhibition; above this value ATP inhibited hexokinase in the presence of glucose 6-phosphate. ATP had an inhibitory effect on soluble hexokinase similar to that on a whole-homogenate hexokinase, so that the ATP inhibition could not be explained by a conversion of particulate into soluble hexokinase (which is more sensitive to inhibition by glucose 6-phosphate). It is concluded that ATP potentiates glucose 6-phosphate inhibition of cerebral-cortex hexokinase, whereas the ATP-Mg(2+) complex has no effect. Inorganic phosphate and l-alpha-glycerophosphate relieved glucose 6-phosphate inhibition of hexokinase; these effects could not be explained by changes in the concentration of glucose 6-phosphate during the assay. 5. The inhibition of hexokinase by ADP appeared to be independent of the glucose 6-phosphate effect and was not relieved by inorganic phosphate. 6. The physiological significance of the ATP, inorganic phosphate and alpha-glycerophosphate effects is discussed in relation to the control of glycolysis in cerebral-cortex tissue.     

Glucose 6-phosphate-dependent binding of hexokinase to membranes of ascites tumor cells.

A pH-dependent, saturable binding of hexokinase isozyme I from Ehrlich ascites carcinoma to plasma membrane and microsome preparations from the same tissue is demonstrated. This binding is enhanced by glucose 6-phosphate and may be considered as the sum of a glucose 6-phosphate-dependent binding and an independent binding. The half saturation concentration of hexokinase is about 0.4 unit per ml for both types of binding, and a maximal binding of 0.5-2.0 units per mg membrane protein is observed for both, although the pH optimum of the independent binding (5.4) is lower than that of the dependent binding (5.9). The half saturation concentration of glucose 6-phosphate required for the dependent binding is 0.05 mM at pH 6.1. 2-Deoxyglucose 6-phosphate competatively reverses the effect of glucose 6-phosphate on binding but does not diminish its inhibition of hexokinase activity.     

Uncoupling of the glucose growth defect and the deregulation of glycolysis in Saccharomyces cerevisiae Tps1 mutants expressing trehalose-6-phosphate-insensitive hexokinase from Schizosaccharomyces pombe

In the yeast Saccharomyces cerevisiae inactivation of trehalose-6-phosphate (Tre6P) synthase (Tps1) encoded by the TPS1 gene causes a specific growth defect in the presence of glucose in the medium. The growth inhibition is associated with deregulation of the initial part of glycolysis. Sugar phosphates, especially fructose-1,6-bisphosphate (Fru1,6bisP), hyperaccumulate while the levels of ATP, Pi and downstream metabolites are rapidly depleted. This was suggested to be due to the absence of Tre6P inhibition on hexokinase. Here we show that overexpression of Tre6P (as well as glucose-6-phosphate (Glu6P))-insensitive hexokinase from Schizosaccharomyces pombe in a wild-type strain does not affect growth on glucose but still transiently enhances initial sugar phosphate accumulation. We have in addition replaced the three endogenous glucose kinases of S. cerevisiae by the Tre6P-insensitive hexokinase from S. pombe. High hexokinase activity was measured in cell extracts and growth on glucose was somewhat reduced compared to an S. cerevisiae wild-type strain but expression of the Tre6P-insensitive S. pombe hexokinase never caused the typical tps1Delta phenotype. Moreover, deletion of TPS1 in this strain expressing only the Tre6P-insensitive S. pombe hexokinase still resulted in a severe drop in growth capacity on glucose as well as sensitivity to millimolar glucose levels in the presence of excess galactose. In this case, poor growth on glucose was associated with reduced rather than enhanced glucose influx into glycolysis. Initial glucose transport was not affected. Apparently, deletion of TPS1 causes reduced activity of the S. pombe hexokinase in vivo. Our results show that Tre6P inhibition of hexokinase is not the major mechanism by which Tps1 controls the influx of glucose into glycolysis or the capacity to grow on glucose. In addition, they show that a Tre6P-insensitive hexokinase can still be controlled by Tps1 in vivo.     

Dissociation and catalysis in yeast hexokinase A.

1. The specific activity of yeast hexokinase A depends on the concentration of the protein in the solution being assayed. When a solution containing 13.5 mg of hexokinase A/ml is diluted 10--100-fold at various values of pH and temperature, there is a gradual decline in the specific activity of the enzyme until an equilibrium value is reached, which varies with the chosen experimental conditions. 2. The catalytic activity lost when hexokinase A (1 mg/ml) is incubated at 30degreesC is recovered by lowering the temperature to 25degreesC. 3. These concentration- and temperature-dependent phenomena are consistent with the existence of a monomer-dimer equilibrium in which the dimer alone is the catalytic form of the enzyme. 4. Glucose alone prevents the decline in specific activity of hexokinase A after dilution, but it does not re-activate dilute solutions solutions of the enzyme. It is concluded that glucose binds to both the dimer and the monomer and prevents both association and dissociation. 5. The progress curve describing the phosphorylation of glucose catalysed by hexokinase A does not attain a steady state. It is possible that dissociation of catalytically active dimers in a ternary complex with glucose and ATP (or glucose 6-phosphate and ADP) could explain the non-linearity of this progress curve.     

Isolation and characterization of yeast protease B.

Protease B was purified from baker's yeast. The final preparation appeared homogeneous by ultracentrifugation and electrophoresis. The S₂₀, ω value of the enzyme was 3.1 S and its molecular weight was calculated to be 31,000 from the results of sedimentation equilibrium analysis. The amino acid composition of the enzyme was also investigated. The enzyme inactivates phosphogluconate dehydrogenase and uricase, but not malate dehydrogenase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase or hexokinase.     

Characterisation of hexokinase in Toxoplasma gondii tachyzoites

We have cloned the hexokinase [E.C. 2.7.1.1] gene of Toxoplasma gondii tachyzoite and obtained an active recombinant enzyme with a calculated molecular mass of 51,465Da and an isoelectric point of 5.82. Southern blot analysis indicated that the hexokinase gene existed as a single copy in the tachyzoites of T. gondii. The sequence of T. gondii hexokinase exhibited the highest identity (44%) to that of Plasmodium falciparum hexokinase and lower identity of less than 35% to those of hexokinases from other organisms. The specific activity of the homogeneously purified recombinant enzyme was 4.04 micromol/mg protein/min at 37 degrees C under optimal conditions. The enzyme could use glucose, fructose, and mannose as substrates, though it preferred glucose. Adenosine triphosphate was exclusively the most effective phosphorus donor, and pyrophosphate did not serve as a substrate. K(m) values for glucose and adenosine triphosphate were 8.0+/-0.8 microM and 1.05+/-0.25mM, respectively. No allosteric effect of substrates was observed, and the products, glucose 6-phosphate and adenosine diphosphate, had no inhibitory effect on T. gondii hexokinase activity. Other phosphorylated hexoses, fructose 6-phosphate, trehalose 6-phosphate which is an inhibitor of yeast hexokinase, and pyrophosphate, also did not affect T. gondii hexokinase activity. Native hexokinase activity was recovered in both the cytosol and membrane fractions of the whole lysate of T. gondii tachyzoites. This result suggests that T. gondii hexokinase weakly associates with the membrane or particulate fraction of the tachyzoite cell.     

The binding of 25-hydroxycholecalciferol and 25-hydroxyergocalciferol to receptor proteins in a New World and an Old World primate.

1. Dirofilaria immitis hexokinase was relatively heat stable and had a pH optimum range between 7.8 and 8.2. 2. Mean Vmax was 0.40 +/- 0.10 (S.D.) mumole/min/100 mg of worm. 3. Mean Km values were 0.32 mM for glucose, 0.86 mM for fructose and 0.39 mM for ATP. 4. Glucose-6-phosphate was not a strong product inhibitor. 5. Starch gel electrophoresis demonstrated at least three isozymes.     

THE OVEREXPRESSED HEXAHISTIDINE-TAGGED HUMAN HEXOKINASE TYPE III IS INHIBITED BY D-GLUCOSE

ABSTRACT

Inhibition by its product, glucose, is a kinetic property of hexokinase type III. In this paper, we report the overexpression in Escherichia coli of human hexokinase type III. The recombinant enzyme was genetically fused with a hexahistidine peptide at the C-terminal end. This modification confers to the product the ability to bind the Ni²⁺ ion immobilised into agarose by nitrilotriacetic acid (NTA) groups. The purification was performed by one-step column chromatography using ammonium sulphate as stabilising agent.

Recombinant hexokinase type III appears as a single band of approximately 100 kDa on a SDS-PAGE gel and shows specific activity of 16 U/mg. Its kinetic parameters are comparable to those of the native enzyme, including the fact that it can be inhibited by glucose. The comparison of these results with the properties of the overexpressed carboxyl-domain led us to suppose that the inhibition site for glucose required the presence of the N-terminal domain.     

A large-scale purification of recombinant histone H1.5 from Escherichia coli

The conversion of glucose into glucose 6-phosphate (Glc 6-P)1 traps glucose in a chemical state in which it cannot leave the cell and hence commits glucose to metabolism. In human tissues there are at least three hexokinase isoenzymes responsible for hexose phosphorylation. These enzymes are constituted by a single polypeptide chain with a molecular weight of approximately 100 kDa. Among these isoenzymes, hexokinase type I is the most widely expressed in mammalian tissues and shows reversion of Glc 6-P inhibition by physiological levels of inorganic phosphate. In this work the hexokinase I from human brain was overexpressed in Escherichia coli, as a hexahistidine-tagged protein with the tag extending the C-terminal end. An average of 900 U per liter of culture was obtained. The expressed protein was one-step purified by metal chelate affinity chromatography performed in NTA-agarose column charged with Ni(2+) ions. In order to stabilize the enzymatic activity 0.5 M ammonium sulfate was added to elution buffer. The specific activity of purified hexokinase I was 67.8 U/mg. The recombinant enzyme shows kinetic properties in agreement with those described for the native enzyme, and thus it can be used for biophysical and biochemical investigation.     

The isolation and characterization of mouse liver glyoxalase I.

The purification of glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing) EC 4.4.1.5) from DBA/1J mouse liver employing ion exchange and affinity chromatography is described. The enzyme was purified 1140-fold and it exhibits a specific activity of 2200 units/mg of protein. The activity was determined to be homogeneous by sedimentation velocity and sedimentation equilibrium ultracentrifugation and by polyacrylamide electrophoresis. The molecular weight is approimately 43 000 and the sedimentation coefficient is 3.4 S. Kinetic data are consistent with a one-substrate (hemimercaptal) reaction mechanism but do not rule out alternate branches at low substrate and free glutathione concentrations.     

The interaction of phosphorylated sugars with human hexokinase I

Glucose 6-phosphate as well as several other hexose mono- and diphosphates were found by kinetic studies to be competitive inhibitors of human hexokinase I (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) versus MgATP. Limited proteolysis by trypsin does not destroy the hexokinase activity but produces as well-defined peptide map when the digested enzyme is electrophoresed in the presence of sodium dodecyl sulfate. MgATP at subsaturating concentration protects hexokinase from trypsin digestion, while phosphorylated sugars, Mg2+, glucose and inorganic phosphate have no effect. Addition of glucose 6-phosphate to the MgATP-hexokinase complex at a concentration 100-times higher than its Ki was not able to reverse the MgATP-induced conformation of hexokinase, suggesting that the binding of glucose 6-phosphate and MgATP are not mutually exclusive. Similar evidence was also obtained by studies of the induced modifications of ultraviolet spectra of hexokinase by the binding of MgATP, glucose 6-phosphate and both compounds. Among a library of monoclonal antibodies produced against rat brain hexokinase I and that recognize human placenta hexokinase I, one (4A6) was found to be able to modify the Ki of glucose 6-phosphate (from 25 to 140 microM) for human hexokinase I. The same antibody also weakens the inhibition by all the other hexoses phosphate studied without affecting the apparent Km for MgATP (from 0.6 to 0.75 mM) or for glucose. These data support the view for the binding of glucose 6-phosphate at a regulatory site on the enzyme.     

Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII

Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression. Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants. The hybrid constructs allowed the identification of a domain (amino acid residues 102-246) associated with the fructose/glucose phosphorylation ratio. This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII. Glucose repression was associated with the C-terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active. Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase. These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain. Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII. This proved that hexokinase PI also has some function in glucose repression. Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII. This showed that hexokinase PI is also capable of mediating glucose repression. All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression. To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced. Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose. Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression. To verify the role of hexokinases in glucose repression, the third glucose-phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double-null mutant. No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose-phosphorylating activity, which is threefold increased compared to wild-type cells. This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.