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PubMedDate not supplied

[Determination of hexokinase isoenzymes in different tissues].

A modified method is described for estimation of hexokinase isoenzymes by polyacrylamide gel electrophoresis. The best conditions for detection of hexokinase activity were developed using immobilization of glucose-6-phosphate dehydrogenas: in gel, utilization of riboflavin as a catalyst of polymerization and stabilization by glucose. The method is simple, highly reproducible, comparatively rapid and economically reasonable.

Electrophoresis, Polyacrylamide Gel↗
PubMed1995

Comparison between the Bayer ELITE glucometer and the hexokinase method for glucose determination on the Eppendorf EPOS 5060 using capillary whole blood samples.

The ELITE glucometer (Bayer Diagnostics), which uses a glucose oxidase sensor coupled to an amperometric measurement, was compared with the routine hexokinase method on the EPOS (Eppendorf--Netheler-Hinz) using commercially available reagents (Boehringer-Mannheim). Both methods were carried out using whole blood as sample. The comparison was made under routine conditions on 106 non-selected patients from whom a glucose determination had been requested. The results gave rise to a correlation coefficient of 0.986 with the regression equation: GlucoseELITE = 1.04 x GlucoseEPOS -0.201 mmol/l. Deviations between both methods were seen, although these had little or no clinical relevance. A positive deviation in favour of the ELITE was seen in a blood sample with 21.8 mmol/l glucose (EPOS) and 1025 mumol/l uric acid. A negative deviation, with respect to the ELITE, was seen in a blood sample with 3.00 mmol/l glucose (EPOS). Blood ethanol concentration up to 0.1 mol/l and uric acid concentrations up to 450 mumol/l did not directly influence the ELITE method. The intra-assay coefficient of variation of the ELITE glucometer was 5.3% at 4.95 mmol/l and 3.1% at 15.2 mmol/l. The corresponding inter-assay coefficients of variation were 6.8% at 5.14 mmol/l and 3.90% at 15.4 mmol/l. Both sets of results were derived from 20 measurements made consecutively or on 20 consecutive days. The ELITE glucometer proved to be a hygienic and practical alternative for on-site measurement of blood glucose and gave results comparable with those using the routine hexokinase method with haemolysed capillary blood.

Blood Chemical Analysis↗
PubMedDate not supplied

[Several structural aspects of the interaction of yeast hexokinase with insulin].

It has been noted that due to formation of the complex with insuline light and hear resistance of hexokinase from baker's yeast rises. Variation of luminescence parameters shows structural modification of enzyme upon complex formation. On the basis of comparison the photoinactivation data and hexokinase photolysis conclusion has been drawn that the tryptophanyl residues are not directly involved in the enzyme active site, although play an important role in supporting the specific structure.

Catalysis↗
PubMed1994

[Analysis of glucose metabolism in patients with esophageal cancer by PET: estimation of hexokinase activity in the tumor and usefulness for clinical assessment using 18F-fluorodeoxyglucose].

We evaluated glucose metabolism of esophageal cancer by PET using 18F-fluorodeoxyglucose (FDG), in order to investigate its clinical usefulness. In 11 advanced cases k3 value reflecting hexokinase activity and Ci/Cp ratio expressing FDG uptake were calculated from radioactivity in the tumor (Ci) and the plasma (Cp). Both k3 and Ci/Cp were well correlated with hexokinase activity from the resected specimen, so Ci/Cp was considered to be a convenient index for clinical assessment of esophageal cancer. Forty-two cases before treatment revealed high accumulation of FDG, and 41 showed more than 2.0 of Ci/Cp. But, 10 normal controls and one esophageal benign tumor showed less than 2.0. As for 13 post-operative cases, 6 cases out of 7 with recurrence showed more than 2.0 for Ci/Cp, but all 6 cases of non-recurrent cases showed less than 2.0. FDG PET is an useful tool for differential diagnosis of recurrence. The clinicopathological findings were investigated in 26 resected cases. Age, location, vertical extension, histologic feature, lymph node metastasis and histologic stage were not correlative with Ci/Cp. As for DNA ploidy pattern, aneuploidy group showed significantly high Ci/Cp rather than diploidy group. Eight cases which showed more than 5.0 of Ci/Cp resulted in poor prognoses compared with 14 cases which showed less than 5.0.

Aged↗
PubMedDate not supplied

[The cooperative effect of hydrocortisone, adrenaline, and high density lipoproteins in regulating the activity of multiple forms of liver hexokinase].

High density lipoproteins (HDL) were shown to modulate the effect of hydrocortisone and adrenaline on activity of hexokinase in surviving liver tissue slices. The hormones inhibited the enzymatic activity while in presence of HDL the hormones exhibited an opposite effect. The phenomenon observed was termed a "cooperative effect"; it could not be reproduced by any single components of the system studied. Mechanism of the effect involved induction of all the multiple hexokinase forms which was abolished by actinomycin D. A specific function could be ascribed only to HDL3; HDL2, low and very low density lipoproteins did not exhibit any effects. The cooperative effect of HDL and of the adaptive hormones was also inhibited in the surviving liver tissue slices by vinblastine, gordox and methylamine thus demonstrating the sophisticated nature of the mechanism.

Animals↗
PubMedDate not supplied

[Effect of alpha-glycerophosphate on intracellular distribution of hexokinase in the tissue of fetal rabbits during acute hypoxia].

In experiments with 40 rabbit fetuses acute intrauterine hypoxia caused a redistribution of the hexokinase activity in brain and liver tissues towards an increase in the amount of the enzyme associated with mitochondria. Previous administration of alpha-glycerophosphate into rabbit female (at a dose of 1.3 g/kg of body weight) was accompanied by an increase in the hexokinase activity in liver mitochondria and in brain cytoplasmic fraction of fetus as well as in the total homogenate of both tissue. The phenomenon might be considered as a positive pattern in adaptation to hypoxia.

Acute Disease↗
PubMedDate not supplied

[Properties of free and bound glycolytic enzymes from the scorpionfish brain. I. Hexokinase].

It was many times shown that hexokinase is tightly bound to cellular structural elements form. However, the comparative analysis of free and bound forms properties in the fish tissues, in particular in brain, has not been done. This analysis was the aim of our experiments with sea scorpion fish brain. Kinetic properties, pH and short-time heating influence on the free and bound hexokinase activity are described in the paper. Possible physiological role of the both enzymes forms is discussed.

Animals↗
PubMed1997

Experimental evidence for dynamic compartmentation of ADP at the mitochondrial periphery: coupling of mitochondrial adenylate kinase and mitochondrial hexokinase with oxidative phosphorylation under conditions mimicking the intracellular colloid osmotic pressure.

Dextran M20 was added to isolated rat liver mitochondria to mimic cytosolic macromolecules. Under these conditions, the morphological changes in the mitochondrial periphery that occur upon isolation of the organelle are restored, i.e. the volume of the intermembrane space decreases and the contact site frequency increases. The ADP routing from mitochondrial kinases at various locations was investigated by using the activities of oxidative phosphorylation and externally added pyruvate kinase as sensors for ADP transport into the matrix and extramitochondrial compartment, respectively. The studies reveal that a significant fraction of the ADP generated by either adenylate kinase in the intermembrane space or by outer membrane bound hexokinase isozyme I, is not accessible to extramitochondrial pyruvate kinase. Quantitative information on the ADP compartmentation in rat liver mitochondria was obtained by comparing the ADP supply from mitochondrial kinases to oxidative phosphorylation with that of non-bound, extramitochondrially located kinases. This approach allowed us to estimate the ADP diffusion gradients which were present across the outer membrane and between the compartment formed by bound hexokinase and the extramitochondrial compartment. In the presence of 10% dextran M20 these ADP gradients amounted to approximately 12 microM. The possible role of mitochondrial kinases in ADP transport into mitochondria in vivo is discussed.

Adenosine Diphosphate↗
PubMed1997

Effect of experimental diabetes on the activities of hexokinase, glucose-6-phosphate dehydrogenase and catecholamines in rat erythrocytes of different ages.

Hexokinase and glucose-6-phosphate dehydrogenase were assayed in various circulating age fractions i.e., young, middle-aged and old red cell from control, diabetic and insulin-treated diabetic rats. An increase in the activity of hexokinase was observed in three age-wise separated fractions of red cells from diabetic animals in comparison to control. The activity of glucose-6-phosphate dehydrogenase on the other hand decreased in separated ageing fractions of diabetic red cells when compared to control. Reversal of these two enzymes were observed in insulin-treated diabetic rats. The levels of glycosylated haemoglobin and catecholamines were found to increase with ageing red cells in controls and also increased in red cells plasma.

Animals↗
PubMed1999

Expression of Human Glucose Transporter Type 1 and Rat Hexokinase Type II Complementary DNAs in Rainbow Trout Embryos: Effects on Glucose Metabolism.

: Sugars are utilized poorly in fish mainly because of low rates of transport across plasma membrane and phosphorylation. To evaluate whether it is possible to augment carbohydrate metabolism in fish using heterologous genes, expression of human glucose transporter type 1 (hGLUT1) and rat hexokinase type II (rHKII) complementary DNAs cloned with cytomegalovirus promoter was followed in rainbow trout embryos. Both genes were transcribed. Hexokinase activity, undetectable in control, was found in transformed blastulas. Increased rates of 14C-methylglucose uptake and sensitivity to cytochalasin B indicated the presence of facilitative hexose transport due to hGLUT1 expression. Effect of hGLUT1 on production of 14CO2 from glucose was greater than that of rHKII. Coexpression of the genes did not increase the rate of glucose oxidation compared with expression of hGLUT1 alone.

Journal Article↗
PubMed2001

Cloning and characterization of a cDNA encoding hexokinase from tomato.

Two different partial sequences encoding putative hexokinase (HXK, ATP: hexose-6-phosphotransferase, EC 2.7.1.1) were isolated from tomato (Lycopersicon esculentum) by RT-PCR using degenerate primers. Southern blot analysis suggested the existence of two divergent HXK genes. A complete cDNA of one HXK was isolated by screening a cDNA library prepared from young cherry tomato fruit. The 1770 bp cDNA of LeHXK2 contained an open reading frame encoding a 496 amino acid protein that has 69% identity with the two Arabidopsis HXKs, 83 and 85% identity with potato StHXK1 and tobacco NtHXK, respectively. However, this clone had 97% amino acid identity with potato StHXK2 and, therefore, was named LeHXK2. LeHXK2 cDNA was expressed in a triple mutant yeast (Saccharomyces cerevisiae) strain which lacked the ability to phosphorylate glucose and fructose and, therefore, was unable to grow on these sugars as carbon sources. Mutant cells expressing LeHXK2 grew on both glucose and fructose with shorter doubling time on glucose. The kinetic properties of LeHXK2 expressed in yeast were determined after the purification of LeHXK2 by HPLC-ion exchange chromatography, confirming the identity of LeHXK2 as hexokinase with higher affinity to glucose. LeHXK2 mRNA was detected by RT-PCR expression analysis in all organs and tissues and at all stages of fruit development. However, semi-quantitative RT-PCR analysis showed that LeHXK2 was most highly expressed in flowers.

Journal Article↗
PubMed1997

Synthetic Utility of Yeast Hexokinase. Substrate Specificity, Cofactor Regeneration, and Product Isolation.

Yeast hexokinase (EC 2.7.1.1) catalyzes the phosphorylation of pyranose and furanose analogs of glucose at 0.01-125% of the rate of glucose. The enzyme is highly tolerant of structural changes at C-2 and C-3 of glucopyranose and less tolerant of changes at C-1 and C-4. Preparative phosphorylations were performed on compounds having 0.01-100% of the activity of glucose, using phosphoenolpyruvate and pyruvate kinase to regenerate ATP. The effects of inhibition of hexokinase by phosphoenolpyruvate and acetyl phosphate on cofactor regeneration are discussed.

Journal Article↗
PubMed1967

Effectors of rat-heart hexokinases and the control of rates of glucose phosphorylation in the perfused rat heart.

1. The kinetic properties of the soluble and particulate hexokinases from rat heart have been investigated. 2. For both forms of the enzyme, the K(m) for glucose was 45mum and the K(m) for ATP 0.5mm. Glucose 6-phosphate was a non-competitive inhibitor with respect to glucose (K(i) 0.16mm for the soluble and 0.33mm for the particulate enzyme) and a mixed inhibitor with respect to ATP (K(i) 80mum for the soluble and 40mum for the particulate enzyme). ADP and AMP were competitive inhibitors with respect to ATP (K(i) for ADP was 0.68mm for the soluble and 0.60mm for the particulate enzyme; K(i) for AMP was 0.37mm for the soluble and 0.16mm for the particulate enzyme). P(i) reversed glucose 6-phosphate inhibition with both forms at 10mm but not at 2mm, with glucose 6-phosphate concentrations of 0.3mm or less for the soluble and 1mm or less for the particulate enzyme. 3. The total activity of hexokinase in normal hearts and in hearts from alloxan-diabetic rats was 21.5mumoles of glucose phosphorylated/min./g. dry wt. of ventricle at 25 degrees . The temperature coefficient Q(10) between 22 degrees and 38.5 degrees was 1.93; the ratio of the soluble to the particulate enzyme was 3:7. 4. The kinetic data have been used to predict rates of glucose phosphorylation in the perfused heart at saturating concentrations of glucose from measured concentrations of ATP, glucose 6-phosphate, ADP and AMP. These have been compared with the rates of glucose phosphorylation measured with precision in a small-volume recirculation perfusion apparatus, which is described. The correlation between predicted and measured rates was highly significant and their ratio was 1.07. 5. These findings are consistent with the control of glucose phosphorylation in the perfused heart by glucose 6-phosphate concentration, subject to certain assumptions that are discussed in detail.

Journal Article↗
PubMed1987

Kinetics of hexokinase D ('glucokinase') with inosine triphosphate as phosphate donor. Loss of kinetic co-operativity with respect to glucose.

When ATP, the normal phosphate donor for hexokinase D ('glucokinase'), is replaced by ITP, the positive co-operativity with respect to glucose disappears. This may be rationalized in relation to kinetic models for hexokinase D co-operativity, which assume that with the normal substrates the chemical reaction and subsequent release of products occur so rapidly that binding of substrates cannot approach equilibrium and is therefore not constrained by the thermodynamic requirement that the Hill coefficient for substrate binding cannot exceed the number of binding sites. ITP is a much poorer substrate than ATP, however: its Km value at high glucose concentrations is 24 times the value for ATP, whereas the value of the limiting rate V is decreased about 8-fold. Consequently it is no longer possible for the ternary complex to be converted into products rapidly enough to generate kinetic co-operativity. The negative co-operativity with respect to glucose observed in 2H2O with ATP as phosphate donor also disappears when ITP is used instead of ATP.

Animals↗
PubMed1994

The Two Km's for ATP of Corn-Root H+-ATPase and the Use of Glucose-6-Phosphate and Hexokinase as an ATP-Regenerating System.

Plasma membrane vesicles derived from corn (Zea mays L.) roots retain a membrane-bound H+-ATPase that is able to form a H+ gradient across the vesicle membranes. The activity of this ATPase is enhanced 2- to 3-fold when Triton X-100 or lysophosphatidylcholine is added to the medium at a protein:detergent ratio of 2:1 (w/w). In the absence of detergent, the ATPase exhibits only one Km for ATP (0.1-0.2 mM), which is the same as for the pumping of H+. After the addition of either Triton X-100 or lysophosphatidylcholine, two Km's for ATP are detected, one in the range of 1 to 3 [mu]M and a second in the range of 0.1 to 0.2 mM. The Vmax of the second Km for ATP increases as the temperature of the assay medium is raised from 15[deg]C to 38[deg]C. The Arrhenius plot reveals a single break at 30[deg]C, both in the absence and in the presence of detergents. In the presence of Triton X-100 the H+-ATPase catalyzes the cleavage of glucose-6-phosphate when both hexokinase and ADP are included in the assay medium. There is no measurable cleavage when the apparent affinity for ATP of the H+-ATPase is not enhanced by Triton X-100 or when 1 mM glucose is included in the assay medium. These data indicate that when the high-affinity Km for ATP is unmasked with the use of detergent, the ATPase can use glucose-6-phosphate and hexokinase as an ATP-regenerating system.

Journal Article↗
PubMed1973

Hexokinase from maize endosperm and scutellum.

Hexokinase (EC 2.7.1.1) was isolated from endosperm and scutellum of developing and germinating maize (Zea mays) seeds. With fructose as the variable substate, Michaelis constant values for the scutellum enzyme were about onethird those of the endosperm enzyme (0.05 versus 0.15 mm), and no developmental differences were observed. With glucose as the variable substrate, Michaelis constant values were all in the range 0.1 to 0.2 mm. The enzyme preparation from germinating scutellum was studied further; when glucose was varied over a wide range, a Michaelis constant of 3.4 mm was observed in addition to the much lower Michaelis constant noted above. This low affinity binding of glucose may have regulatory significance and may indicate the presence of a glucokinase in addition to hexokinase.

Journal Article↗
PubMed1986

Quantitative Measurements of Hexokinase Activity in the Shoot Apical Meristem, Leaf Primordia, and Leaf Tissues of Dianthus chinensis L.

Hexokinase was measured by quantitative histochemical techniques in the apical meristem, primordia, and leaves of Dianthus chinensis L. The structural stages of development in the leaves sampled were determined by light and scanning electron microscopy. The results showed that activity decreased from the youngest primordia (1500 millimoles per kilogram dry weight per hour) to the mature leaves (200 millimoles per kilogram dry weight per hour) and that an intermediate leaf, the fourth youngest, showed the same declining pattern from its base to its tip. Surface views and measurements of these leaves revealed their basipetal maturation as seen by cell size, stomatal development, trichome differentiation, cuticular appearance, and leaf thickness. The intermediate leaf showed features representative of several stages in structural differentiation. It was concluded that the changes in hexokinase activity among the leaves of a shoot and within an individual leaf are similar and correlate with the degree of structural differentiation of the leaves.

Journal Article↗
PubMed1989

Regulation of Glycolytic Flux and Ethanol Production in Saccharomyces cerevisiae: Effects of Intracellular Adenine Nucleotide Concentrations on the In Vitro Activities of Hexokinase, Phosphofructokinase, Phosphoglycerate Kinase, and Pyruvate Kinase.

The progressive decline in the glycolytic activity of Saccharomyces cerevisiae during batch fermentation is accompanied by changes in adenine nucleotide pools. The relative activities of four glycolytic enzymes were examined in vitro in the presence of nucleotide concentrations equivalent to intracellular pools. Phosphofructokinase and pyruvate kinase were not inhibited. Phosphoglycerate kinase was inhibited by AMP but was judged unlikely to be of physiological consequence owing to enzyme abundance. Both isoenzymes of hexokinase were strongly inhibited by AMP. The degree of hexokinase inhibition was sufficient to account for the observed decline in glycolytic activity during batch fermentation.

Journal Article↗