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PubMed1979

Isoenzymes of hexokinase, 6-phosphogluconate dehydrogenase, phosphoglucomutase and lactate dehydrogenase in uterine cancer.

Electrophoresis of cytosol prepared from normal and malignant tissue samples of uterine cervix and endometrium revealed interesting differences which may be relevant to the characteristic alterations in glucose metabolism associated with tumour development. Hexokinase II was detected in 30% of the cancer material from both sources, but in none of the samples of normal cervix. A duplet band of 6-phosphogluconate dehydrognease was seen in the majority of the cancer samples but in no sample of normal cervix; it appeared to be partly due to ageing of the sample, and is not phenotypically related to the malignant process. Analysis of genetic variance for phosphoglucomutase at the PGM1 locus revealed a highly significant excess of the PGM1-1 phenotype in patients with cancer of the endometrium, which may reflect susceptibility to endometrial cancer in patients with this phenotype. At the PGM2 locus, samples of malignant cervix were deficient in "Band f" compared with normal cervix samples, all of which showed this band. Conversely, gene products of the PGM3 locus were found in most samples of malignant cervix and a small minority of normal cervix samples. Compared with the isomorphic distribution of lactate dehydrogenase enzymes in normal uterine tissue, cancers showed a shift towards either a more anodal or a more cathodal pattern. The former may be associated with tumours enjoying a good oxygen supply, and the latter with tumours which, because of their depth or poor blood supply have to function under less aerobic conditions.

Electrophoresis, Polyacrylamide Gel↗
PubMed1996

High glycolysis in gliomas despite low hexokinase transcription and activity correlated to chromosome 10 loss.

Loss of chromosome 10 was observed in 10 out of 12 xenografted glioblastomas studied. Chromosome 10 carries the gene coding the hexokinase type 1 isoenzyme (HK-I), which catalyses the first step of glycolysis, which is essential in brain tissue and glioblastomas. We investigated the relationships between the relative chromosome 10 number, the amount of HK-I mRNA, HK-I activity and its intracellular distribution, and glycolysis-related parameters such as the lactate-pyruvate ratio, lactate dehydrogenase (LDH) and ATP contents. Individual tumour HK-I mRNA amounts were 23-65% lower than that of normal human brain and reflected the relative decrease of chromosome 10 number (alpha < 0.01). Total HK activities of individual glioblastomas varied considerably but were constantly (a mean of seven times) lower than that of normal brain tissue. The mitochondria-bound HK-I fraction of individual tumours was generally over 50%, compared with that of normal brain tissue. As shown by lactate - pyruvate ratios, in all the gliomas, glycolysis was elevated to an average of 3-fold that measured in normal brain. An elevated ATP content was also constantly noted. Adaptation of glioblastoma metabolism to the chromosome 10 loss and to the HK-I transcription unit emphasises the critical role of glycolysis in their survival. We hypothesise that HK-I, the enzyme responsible for initiating glycolysis necessary for brain function, may approach its lowest limit in gliomas, thereby opening therapeutic access to pharmacological anti-metabolites affecting energy metabolism and tumour growth.

Adenosine Triphosphate↗
PubMed2006

Hexokinase II: cancer's double-edged sword acting as both facilitator and gatekeeper of malignancy when bound to mitochondria.

A key hallmark of many cancers, particularly the most aggressive, is the capacity to metabolize glucose at an elevated rate, a phenotype detected clinically using positron emission tomography (PET). This phenotype provides cancer cells, including those that participate in metastasis, a distinct competitive edge over normal cells. Specifically, after rapid entry of glucose into cancer cells on the glucose transporter, the highly glycolytic phenotype is supported by hexokinase (primarily HK II) that is overexpressed and bound to the outer mitochondrial membrane via the porin-like protein voltage-dependent anion channel (VDAC). This protein and the adenine nucleotide transporter move ATP, newly synthesized by the inner membrane located ATP synthase, to active sites on HK II. The abundant amounts of HK II bind both the ATP and the incoming glucose producing the product glucose-6-phosphate, also at an elevated rate. This critical metabolite then serves both as a biosynthetic precursor to support cell proliferation and as a precursor for lactic acid, the latter exiting cancer cells causing an unfavorable environment for normal cells. Although helping facilitate this chemical warfare, HK II via its mitochondrial location also suppresses the death of cancer cells, thus increasing their possibility for metastasis and the ultimate death of the human host. For these reasons, targeting this key enzyme is currently being investigated in several laboratories in a strategy to develop novel therapies that may turn the tide on the continuing struggle to find effective cures for cancer. One such candidate is 3-bromopyruvate that has been shown recently to eradicate advanced stage, PET positive hepatocellular carcinomas in an animal model without apparent harm to the animals.

Adenine Nucleotides↗
PubMed1967

The subcellular distribution and properties of hexokinases in the guinea-pig cerebral cortex.

1. Hexokinase activities were estimated in primary subcellular fractions from guinea-pig cerebral cortex and in sucrose-density-gradient subfractions of the mitochondrial and microsomal fractions. 2. Appreciable activities were observed in mitochondrial, microsomal and soluble fractions. The activity in the mitochondrial fraction was associated with the mitochondria rather than with myelin or nerve endings and that in the microsomal fraction was associated with membrane fragments. 3. Most of the mitochondrial activity was extracted in soluble form by osmotic ;shock'. The activity of the mitochondrial extract differed from the soluble activity in kinetic properties and in electrophoretic behaviour. 4. No evidence was obtained for the presence of a high-K(m) glucokinase in the brain. 5. The results are discussed in terms of relevance to considerations of glucose utilization by the brain.

Adenosine Triphosphate↗
PubMed1971

Allosteric activation of brain hexokinase by magnesium ions and by magnesium ion--adenosine triphosphate complex.

1. Substrate-saturation curves of brain hexokinase for MgATP(2-) were sigmoidal at sub-saturating concentrations of glucose when the Mg(2+)/ATP ratio was maintained at 1:1. Under identical conditions, except that Mg(2+) was present in excess, hyperbolic curves were observed. 2. The number of binding sites (calculated from Hill plots) is 1.8 at a Mg(2+)/ATP ratio 1:1, and 1.0 with excess of Mg(2+). The apparent K(m) for MgATP(2-) is 6.5x10(-4)m at a Mg(2+)/ATP ratio 1:1, and 3.5x10(-4)m with excess of Mg(2+). 3. Interdependence between substrate-binding sites was indicated by the effects of varying the concentration of glucose. The sigmoidality and deviation from Michaelis-Menten kinetics at a Mg(2+)/ATP ratio 1:1 became less pronounced with increasing glucose concentration. Also, although substrate-saturation curves for glucose were hyperbolic when the Mg(2+)/ATP ratio was 1:1, reciprocal plots were non-linear. These were linear with excess of Mg(2+). 4. High concentrations of Mg(2+) (Mg(2+)/ATP ratios above 5:1) were inhibitory. 5. The results are taken to indicate homotropic co-operative binding of MgATP(2-) and that Mg(2+) is an allosteric activator. Possible implications in regulation are discussed.

Adenosine Triphosphate↗
PubMed1972

The use of deoxyfluoro-D-glucopyranoses and related compounds in a study of yeast hexokinase specificity.

1. 2-Deoxy-2-fluoro-d-glucose, 2-deoxy-2-fluoro-d-mannose and 2-deoxy-2,2-difluoro-d-arabino-hexose are good substrates for yeast hexokinase. 2. 3-Deoxy-3-fluoro-d-glucose and 4-deoxy-4-fluoro-d-glucose are poor substrates and have very similar K(m) values (8x10(-2)m). 3. Neither alpha- nor beta-d-glucopyranosyl fluoride is a substrate or inhibitor. 4. Studies with 2-chloro-2-deoxy- and 2-O-methyl derivatives of d-glucose and d-mannose have revealed that little chemical modification is possible at position 2 without substantial loss in substrate binding. 5. The variation in the value of K(m) for the d-hexose derivatives was associated with a corresponding change in the value of K(m) for MgATP(2-) showing that the binding of MgATP(2-) is modified by the binding of the sugar.

Adenosine Triphosphate↗
PubMed1972

Activation of brain hexokinase by magnesium ions and by magnesium ion--adenosine triphosphate complex.

1. An alternative explanation for the kinetic data obtained by Bachelard (1971) for the brain hexokinase reaction is presented. 2. Apparently sigmoidal saturation curves for MgATP(2-) based upon Bachelard's (1971) studies can be corrected to hyperbolic curves by use of a stability constant for MgATP(2-) complex formation. 3. A number of other effects related to the concentration-dependent stability of the MgATP(2-) complex and to the presence of the inhibitory free uncomplexed ATP(4-) concentration are also explained in terms of a non-allosteric role for either Mg(2+) or MgATP(2-) fully consistent with a number of previous reports on this enzyme. 4. A brief discussion of the validity of Hill plots in studies of multisubstrate co-operative enzymes is presented. 5. A simple model is presented that demonstrates how enzymes obeying Michaelis-Menten kinetics can demonstrate sigmoidal velocity responses if the true substrate of the reaction is the metal-substrate complex.

Adenosine Triphosphate↗
PubMed1974

Difference in kinetic properties between hexokinase type I isoenzymes from various rat tissues with reference to the effect of a thiol inhibitor.

Hexokinase isoenzyme type I was purified from various rat tissues, and was subjected to kinetic studies in the presence or the absence of p-chloromercuribenzenesulphonate. The mode of the inhibition by the thiol inhibitor was different for the type I isoenzymes obtained from different tissues, suggesting that the type I isoenzymes from different tissues were not identical proteins.

Adenosine Triphosphate↗
PubMed1977

The catalytic activity of monomeric yeast hexokinase A.

It has been suggested [Williams, D.C. & Jones, J.G. (1976) Biochem. J. 155, 661-667] that monomeric hexokinase isoenzyme A is not catalytically active. We here present data from reacting-enzyme sedimentation, dissociation experiments and from previous studies which are consistent with the monomeric form possessing catalytic activity.

Catalysis↗
PubMed1979

The effect of restricted hydration on the rate of reaction of glucose 6-phosphate dehydrogenase, phosphoglucose isomerase, hexokinase and fumarase.

A method is described for the measurement of enzyme activity under xeric conditions. The reaction mixtures had water contents ranging between 0.1 and 0.6g/g of reaction mixture. For glucose 6-phosphate dehydrogenase, hexokinase and fumarase, enzyme activity became detectable (about 0.05% of the fully hydrated rate) when the water content was about 0.2g/g of reaction mixture, and for phosphoglucose isomerase, around 0.15g/g of reaction mixture. With the water content raised to 0.3g/g of reaction mixture the reaction rates were only increased to 0.1-3% of the fully hydrated rate. When the combined rates for phosphoglucose isomerase and glucose 6-phosphate dehydrogenase were measured, reasonable agreement was found between the experimental data and those calculated from the individual experimentally determined rates on the assumption that diffusion was not further limiting. A method was devised for measuring the diffusion coefficients of low-molecular-weight substances in solutions having low water contents. The diffusion coefficients of riboflavin in sorbitol solution decreased by about 100-fold when the water content of the latter was reduced from 3 to 0.25g/g of sorbitol. It is concluded that to detect enzyme activity a certain minimal amount of water is required and that above this minimum the rate is still restricted by diffusion limitation. The relevance of the results to the physical state of water in reaction mixtures and to metabolism in seeds and spores in xeric conditions is discussed.

Diffusion↗
PubMed1981

The stereochemical course of yeast hexokinase-catalysed phosphoryl transfer by using adenosine 5'[gamma(S)-16O,17O,18O]triphosphate as substrate.

Adenosine 5'[gamma(S)-16O, 17O, 18O]triphosphate has been synthesized and used to determine the stereochemical course of phosphoryl transfer catalysed by yeast hexokinase. The chirality at phosphorus of the D-glucose 6-[16O,17O,18O]phosphate formed was analysed, after cyclization and methylation, by 31P n.m.r. spectroscopy. The phosphoryl transfer was found to occur with inversion of configuration, with a stereoselectivity in excess of 94%. The simplest interpretation of this result is that the phosphoryl group is transferred between substrates in the enzyme-substrate ternary complex by an 'in line' mechanism.

Adenosine Triphosphate↗
PubMed2003

Control of glucose phosphorylation in L6 myotubes by compartmentalization, hexokinase, and glucose transport.

In muscle, insulin enhances influx of glucose and its conversion to glucose 6-phosphate (G6P) by hexokinase (HK). While effects of insulin on glucose transport have been demonstrated, its effect on the activity of HK of cells has not. In L6 myotubes treated for 24 h with insulin there was increased expression of the HK isoform, HKII, and increased glucose phosphorylation without a concomitant increase in glucose transport, indirectly suggesting that phosphorylation of glucose was a target of insulin action [Osawa, Printz, Whitesell and Granner (1995) Diabetes 44, 1426-1432]. In the present work the same treatment led to a 2-fold rise in G6P, suggesting that transport and/or HK were important targets of insulin action. We used a method to identify the site of rate control involving the specificity of phosphorylation towards 2-deoxy-[1-14C]glucose and D-[2-3H]glucose. Glucose transport does not greatly discriminate between these two tracers while HK shows increased specificity for glucose. Specificity of the glucose phosphorylation of the cells increased with addition of insulin and when extracellular glucose was raised. Specificity was reduced at low glucose concentrations or when the inhibitor of transport, cytochalasin B, was added. We conclude that transport and HK share nearly equal control over glucose phosphorylation in these cells. A computer program was used to test models for compatibility with the different types of experiments. The predicted intracellular glucose and transport rates associated with phosphorylation activity were lower than their measured values for the whole cell. In the most likely model, 15+/-4% of the glucose transporters serve a proportionate volume of the cytoplasm. Insulin activation of glucose phosphorylation might then result from stimulation of these transporters together with HK recruitment or relief from inhibition by G6P.

Biological Transport↗
PubMed2004

In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death.

In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating. The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood. Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis. In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time. Moreover, HK-I prevented the Ca(2+)-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c. The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways. Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I. The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I. Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death. These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism.

Animals↗
PubMed1985

Anomeric specificity of hexokinase and glucokinase activities in liver and insulin-producing cells.

Conflicting data have been reported concerning the anomeric specificity of glucokinase. In the present study, liver hexokinase (Km for D-glucose 0.4 mM) displayed a higher affinity for but lower Vmax. with alpha- than with beta-D-glucose. The velocity of the reaction catalysed by liver glucokinase was higher with with beta- than with alpha-D-glucose, whatever the glucose concentration. The apparent Km of glucokinase was somewhat lower, however, with alpha- than with beta-D-glucose. Comparable results were obtained for the high-Km glucokinase-like enzymic activity present in normal pancreatic islets or insulin-producing tumoral cells. These results suggest that the anomeric specificity of glucokinase cannot account for the higher rate of glycolysis found in islets exposed to alpha- as distinct from beta-D-glucose.

Animals↗
PubMed1987

Kinetic properties of hexokinase as assembled with a microcomputer data base.

We have constructed a relational data base containing the kinetic properties of the isoenzymes of hexokinase using the Knowledgeman data-base program on an IBM PC microcomputer. The natural subunit of this data base is the refereed publication, 165 of which were included. Reported values for the Mr (approx. 97,000) are in good agreement, but this agreement becomes progressively worse as one examines the Km values for glucose and ATP and the Ki for glucose 6-phosphate, where the reported values are spread over three orders of magnitude. Some quantities are very thinly or unreliably determined. Experimental conditions, especially free Mg2+ concentration, are rarely close to physiological. Reasons for the spread or uncertainty of numbers, and the distinctions that can be made between isoenzymes despite this spread, are discussed.

Adenosine Triphosphate↗
PubMed1989

An artificial-intelligence technique for qualitatively deriving enzyme kinetic mechanisms from initial-velocity measurements and its application to hexokinase.

We have developed a computer method based on artificial-intelligence techniques for qualitatively analysing steady-state initial-velocity enzyme kinetic data. We have applied our system to experiments on hexokinase from a variety of sources: yeast, ascites and muscle. Our system accepts qualitative stylized descriptions of experimental data, infers constraints from the observed data behaviour and then compares the experimentally inferred constraints with corresponding theoretical model-based constraints. It is desirable to have large data sets which include the results of a variety of experiments. Human intervention is needed to interpret non-kinetic information, differences in conditions, etc. Different strategies were used by the several experimenters whose data was studied to formulate mechanisms for their enzyme preparations, including different methods (product inhibitors or alternate substrates), different experimental protocols (monitoring enzyme activity differently), or different experimental conditions (temperature, pH or ionic strength). The different ordered and rapid-equilibrium mechanisms proposed by these experimenters were generally consistent with their data. On comparing the constraints derived from the several experimental data sets, they are found to be in much less disagreement than the mechanisms published, and some of the disagreement can be ascribed to different experimental conditions (especially ionic strength).

Adenosine Triphosphate↗
PubMed1995

Kinetics of the cooperative binding of glucose to dimeric yeast hexokinase P-I.

Kinetic studies of the cooperative binding of glucose to yeast hexokinase P-I at pH 6.5 have been carried out using the fluorescence temperature-jump technique. Three relaxation effects were observed: a fast low-amplitude effect which could only be resolved at low glucose concentrations (tau 1(-1) = 500-800 s-1), an intermediate effect (tau 2) which showed a linear dependence of reciprocal relaxation time on concentration, and a slow effect (tau 3) which showed a curved dependence on glucose concentration, increasing from approximately 28 s-1 at low concentrations to 250 s-1 at high levels. The findings are interpreted in terms of the concerted Monod-Wyman-Changeux mechanism, the two faster relaxations being assigned to binding to the R and T states, and the slow relaxation to isomerization between the states. Quantitative fitting of the kinetic data to the mechanism has been carried out using independent estimates of the equilibrium parameters of the model; these have been derived from equilibrium dialysis data and by determining the enhancement of the intrinsic ATPase activity of the enzyme by the non-phosphorylatable sugar lyxose, which switches the conformation of the enzyme to the active R state.

Adenosine Triphosphatases↗
PubMed1981

Activities of hexokinase, phosphofructokinase, fructose bisphosphatase and 2-oxoglutarate dehydrogenase in muscle of normal subjects and very ill surgical patients.

1. The activities of hexokinase, phosphofructokinase, fructose bisphosphatase and 2-oxoglutarate dehydrogenase have been measured in the vastus lateralis and rectus abdominus muscle of normal human subjects and in very ill surgical patients. 2. The activities of these enzymes in the muscle of control subjects were similar to the pattern seen in the skeletal muscle of other mammals and lower vertebrates. 3. Fructose bisphosphatase and phosphofructokinase activities were significantly lower in the muscle of ill patients although the depression of the activity of fructose bisphosphatase was much greater than that of phosphofructokinase in both muscle types of ill patients. 4. The maximum rate of cycling in the fructose 6-phosphate--fructose, 1,6-diphosphate cycle may be altered in the ill. 5. This decreased cycling may have a direct influence on the sensitivity of glycolysis to regulators such as the adenine nucleotides and may reduce the ability to maintain body temperature. 6. Increased glycogen synthesis in these muscles may indicate that the role of fructose bisphosphatase is unlikely to be solely in glycogen resynthesis.

Fructose-Bisphosphatase↗