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PubMed1985

The insulin-mimetic action of Mn2+: involvement of cyclic nucleotides and insulin in the regulation of hepatic hexokinase and glucokinase.

Manganese causes a significant rise in hepatic glucokinase and hexokinase in 16-day-old suckling rats, and has an insulinomimetic effect in producing a precocious emergence of glucokinase (EC 2.7.1.2) and a rise in the low Km, hexokinases (EC 2.7.1.1) activities. These enzyme changes occur within 6 hr of manganese administration and there are accompanying increases in plasma insulin and hepatic cyclic GMP. That the effect of manganese is at a site other than, or in addition to, insulin secretion is suggested by the significant increases in glucokinase and hexokinase in 16-day-old streptozotocin-diabetic rats; in this group there is also an increase in hepatic cGMP similar in time scale to that of the normal-manganese-treated group. The effects of manganese and insulin were not additive. It is proposed that one site of action of manganese may be at the level of cyclic GMP systems. The results are also discussed in relation to the known action of manganese at the level of the protein phosphatases.

Animals↗

PubMed1984

The distribution of hexokinase compared to cytochrome oxidase and acetylcholinesterase in the somatosensory cortex and the superior colliculus of the rat.

The distribution of hexokinase, a general glycolytic enzyme, was compared to that of cytochrome oxidase and acetylcholinesterase (AChE) in the somatosensory cortex and the superior colliculus of the rat. The vibrissal barrel fields of the adult rat contain high hexokinase and cytochrome oxidase activity and low AChE activity. In the superior colliculus, hexokinase activity was highest in cell layers and discrete foci of intense activity were observed in the deep grey layer. This distribution was different from that of both cytochrome oxidase and AChE in this structure.

Acetylcholinesterase↗

PubMed1975

An examination of the age-related patterns of decay of the hexokinases of human red cells.

A previous method for red cell fractionation by density gradient centrifugation, using a swing-out rotor, has been scaled up to deal with larger volumes of red cells. This method, involving the use of a zonal rotor, is described and has been applied to the study of the decay of hexokinase in the red cells of normal individuals. Hexokinase activity was seen to fall very rapidly in the young cells followed by a much more gradual decline in older cells. It is estimated that the mature red cell probably contains no more than 2-3% of the hexokinase activity originally present in the reticulocyte. An electrophoretic study showed a changing pattern of the isozymes HK1 and HK2 with increasing cell age. HK2 declines very rapidly in the early fractions whereas HK1 appears to decay more gradually.

Erythrocyte Aging↗

PubMed1996

Crystallization and preliminary X-ray analysis of human brain hexokinase.

Human brain hexokinase type I, expressed in Escherichia coli, has been crystallized from polyethylene glycol 8000 in the presence of inorganic phosphate. The crystals are hexagonal needles of diameter 0.25 mm, diffracting to a resolution of 3.5 A on a rotating-anode/area-detector system. The crystals belong to the space group P3(1)21/P3(2)21 with cell dimensions a = b = 171.5 A and c = 99.4 A. The specific volume of the crystal is 4.2 A3/Da, suggesting an asymmetric unit with a single 100 kDa molecule and a solvent content of 71% by volume or two molecules of hexokinase with a solvent content of 41%. The complex of hexokinase with glucose crystallizes under similar conditions, giving crystals of the same morphology.

Brain↗

PubMed1983

Characterization of Trypanosoma cruzi hexokinase.

Properties of hexokinase (EC 2.7.1.1) from Trypanosoma cruzi epimastigote forms (Tulahuen strain) were studied and compared with enzymes from other sources. The enzyme activity was 37 units g-1 of wet cells (1.2 units mg-1 protein). Hexokinase showed Km values for glucose and ATP of 0.09 and 0.4 mM, respectively. The enzyme reacted with other nucleotides too. N-Acetylglucosamine was a competitive inhibitor with respect to glucose (Ki = 0.3 mM). ADP inhibited the enzyme competitively with respect to ATP (Ki = 1.5 mM) and noncompetitively with respect to glucose (Ki = 7 mM). The enzyme was markedly inhibited by 5-thioglucose, its Ki value was 0.4 mM. Hexokinase activity was not affected by glucose 6-phosphate.

Adenosine Diphosphate↗

PubMed1984

Regulation of energy metabolism in Trypanosoma (Schizotrypanum) cruzi epimastigotes. I. Hexokinase and phosphofructokinase.

Hexokinase (ATP: hexose 6-phosphotransferase, E.C.2.7.1.1) and phosphofructokinase (ATP:fructose-6-phosphate 1-phosphotransferase, E.C.2.7.1.11), two key regulatory enzymes of the glycolytic pathway in vertebrate cells, have been isolated and partially purified from Trypanosoma (Schizotrypanum) cruzi epimastigotes. Both enzymes are associated with particles sedimentable at 105 000 X gav for 1 h and have a high degree of latency; they can be solubilized by sonication. Hexokinase catalyses the phosphorylation of a series of monosaccharides at the following relative rates: D-glucose (100) congruent to D-fructose (97) greater than 2-deoxy-D-glucose (72) congruent to mannose (69) greater than 2-amino-D-glucose (63) greater than 3-O-methyl-D-glucose (21). Very little or no phosphorylating activity was found for D-galactose, N-acetyl-2-amino-D-glucose or 1-alpha-methyl-D-glucose. D-Glucose phosphorylation at fixed ATP concentration follows simple Michaelis-Menten kinetics with Km = 40 microM and Vmax = 440 nmol min-1 mg-1 protein. D-Mannose, 2-deoxy-D-glucose and N-acetyl-2-amino-D-glucose act as competitive inhibitors of glucose phosphorylation, suggesting a single kinase. Mg2+-ATP is the preferred phosphoryl donor, ITP and GTP being much less effective. T. cruzi hexokinase is not inhibited by D-glucose 6-phosphate, or by any of the following compounds (2 mM):D-fructose 6-phosphate, D-fructose 1,6-diphosphate, D-glucose 1,6-diphosphate, phosphoenol pyruvate, L-malate and citrate. Phosphofructokinase displays simple Michaelis-Menten kinetics with no evidence of sigmoidicity with respect to D-fructose 6-phosphate at all ATP concentrations tested, giving a Km of 1.31 mM and Vmax = 400 nmol min-1 mg-1 protein at optimal ATP levels. With respect to ATP, the enzyme exhibits Michaelis-Menten kinetics at low concentration (less than 1 mM) of the substrate (Km = 40 microM at 5 mM MgCl2, pH 7.4). A moderate inhibition is observed at high ATP levels (70% of maximal activity at 2 mM). GTP can substitute for ATP as the phosphoryl donor (Km = 79 microM under the same conditions), but produces only very small inhibitory effects at high concentrations. 5'-AMP activates the enzyme by decreasing its Km with respect to D-fructose 6-phosphate without affecting Vm. Other well-known regulators of the activity of this enzyme in procaryote and vertebrate systems such as citrate, phosphoenol pyruvate, ammonium and phosphate ions have no effect in T. cruzi.

Animals↗

PubMed1987

Isolation and partial characterization of the hexokinase isoenzymes from pathogenic and non-pathogenic strains of Entamoeba histolytica.

Isoenzyme electrophoretic patterns (zymodemes) are increasingly used to distinguish between pathogenic and non-pathogenic strains of Entamoeba histolytica. Isolates of E. histolytica from asymptomatic and symptomatic cases have been shown to differ in the electrophoretic mobility of their hexokinase and phosphoglucomutase isoenzymes. The hexokinase isoenzymes from a non-pathogenic strain and from a pathogenic strain of E. histolytica were purified by fast protein liquid chromatography in several steps, which included a separation by size, chromatofocusing, and anion exchange chromatography. The isoenzymes differed in their isoelectric points, which ranged from pH 4.8-5.4, but had very similar kinetic properties and almost identical apparent molecular weights (48,000) in sodium dodecyl sulfate polyacrylamide gels, as well as on gel filtration columns. Comparison of tryptic peptide analysis of each of the isoenzymes indicated considerable homology between the non-pathogenic and pathogenic forms. Antibodies produced against each of the two pathogenic hexokinase isoenzymes inhibited their enzymatic activity. The antibodies also inhibited the activity of the isoenzymes of the non-pathogenic strain. Our findings suggest that the isoenzymes have structural similarities, and that the pathogenic ones differ from the non-pathogenic ones in their electromobility due to post-translational modifications.

Animals↗

PubMed1995

Nucleotide sequence of the 5'-flanking region of the rat type II hexokinase gene.

In a previous study, we found that the steady state transcript level of type II hexokinase was specifically and remarkably elevated in rat hepatoma AH130 cells. To determine the molecular mechanism of transcriptional control of the type II hexokinase gene, we examined the nucleotide sequence of its 5'-flanking region and analyzed putative transcription factor binding sites. We also examined the type II hexokinase promoter activity by the chloramphenicol acetyltransferase (CAT) assay.

Amino Acid Sequence↗

PubMed1982

A nuclear magnetic relaxation study of conformational changes induced by substrate and temperature in bovine liver thiosulfate sulfurtransferase and yeast hexokinase.

Nuclear magnetic relaxation studies have been performed on thiosulfate sulfurtransferase (EC 2.8.1.1) and hexokinase (EC 2.7.1.1). Observation of proton spin-lattice relaxation times T1 indicates that structural transitions occur in these enzymes in the range 0-40 degrees C and that there are different temperature-dependent forms of thiosulfate sulfurtransferase and hexokinase. Thermal transitions between these forms are affected by the binding of the substrates. The results may be due to changes in the interactions between the structural domains into which the single polypeptide chains of thiosulfate sulfurtransferase and hexokinase are folded.

Animals↗

PubMed1985

Isolation and glucose-6-phosphate-mediated dimerization of hexokinase from human heart.

Soluble hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified from human heart. 1 kg of tissue provided 25 mg hexokinase with a specific activity of 58 units/mg, representing a 1700-fold purification and 47% yield. The purification involved six steps, including affinity chromatography with glucosamine attached to Sepharose. The material was homogeneous according to electrophoresis, gel-filtration and sedimentation in the ultracentrifuge, but gave two main components on electrophoresis in denaturing conditions. From determination of the sedimentation and diffusion coefficients, the relative molecular mass was calculated to be 105 000. The enzyme is monomeric, but glucose 6-phosphate promotes an association to dimers. This effect is reversible and is independent of the concentrations of glucose or inorganic phosphate. The results support the postulate that soluble and mitochondrion-bound hexokinases are identical.

Centrifugation, Density Gradient↗

PubMed1985

A fluorescence study of thermally induced conformational changes in yeast hexokinase.

Fluorescence studies have been performed on yeast hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) as a function of temperature. Observations of both the intrinsic protein fluorescence and the fluorescence of the noncovalently bound apolar probe 2-(p-toluidinyl)naphthalene-6-sulfonic acid under conditions where hexokinase is monomeric, indicate that significant thermal structural transitions occur in the protein over the physiological range of temperature (0 degrees-40 degrees C) and that there are different temperature-dependent forms of the enzyme. Thermal transitions between these forms are affected by the binding of the substrates D-glucose and ATP-Mg. It therefore appears that catalysis connects conformers that differ in stability and the present results are consistent with models in which hexokinase function is linked to changes in the interactions between the domains into which this protein is folded.

Adenosine Triphosphate↗

PubMed1985

Anomeric specificity of mannose phosphorylation by hexokinase.

In rat parotid or pancreatic islet homogenates incubated at 7 degrees C, hexokinase displayed a greater affinity for but a lower maximal velocity with the alpha-anomer, as distinct from beta-anomer, of D-mannose. The anomeric specificity of mammalian hexokinase was similar in the case of D-mannose and D-glucose, but represented a mirror image of that of yeast hexokinase.

Animals↗

PubMed1988

Kinetic regulation of hexokinase activity in a heterogeneous branched bienzyme system.

The kinetic behaviour of a heterogeneous branched bienzyme system of beta-D-glucose oxidase and hexokinase on glucose has been studied. In this sequence, hexokinase is inhibited by its product glucose 6-phosphate and also by D-gluconic acid produced from the parallel enzymic reaction of glucose oxidase. Effect of glucose concentrations on the product's distribution in branched pathway of the bienzyme system is dependent on the kinetic properties of hexokinase and glucose oxidase. Product inhibitions, which are also pH dependent, have a strong regulatory role on the reaction flux.

Gluconates↗

PubMed1991

Decay of mammalian hexokinase: characterization of the specific proteolytic activity.

Hexokinase-degrading activity (HKDA) is associated with hexokinase throughout purification. It is not ubiquitin-dependent. With glucose present, HKDA requires MgATP. It is triggered by removal (or ATP-dependent consumption) of glucose, and masked when glucose is restored. PCMB, TLCK and TPCK suppress hexokinase and activate HKDA. Glucose removal and active-site blockage appear to induce HKDA.

Adenosine Triphosphate↗

PubMed1984

Regulatory properties of rabbit red blood cell hexokinase at conditions close to physiological.

The true level of hexokinase in rabbit erythrocytes was determined by three different methods, including the spectrophotometric glucose-6-phosphate dehydrogenase coupled assay and a new radioisotopic assay. The value found at 37 degrees C (pH 7.2) was 10.23 +/- 1.90 mumol/h per ml red blood cells, which is lower than previously reported values. More than 40 cellular components of the rabbit erythrocytes were tested for their effects on the enzyme. Their intracellular concentrations were also determined. Several of these compounds were found to be competitive inhibitors of the enzyme with respect to Mg X ATP2-. Furthermore, reduced glutathione at a concentration of 1 mM was able to maintain hexokinase in the reduced state with full catalytic activity. The ability of orthophosphate to remove the inhibition of some phosphorylated compounds was examined under conditions similar to cellular (pH 7.2 and 50 microM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP4- and 2,3-diphosphoglycerate to the rabbit hemoglobin significantly modifies their intracellular concentrations and the formation of the respective Mg complexes. The pH-dependence of the reaction velocity and of the kinetic properties of the enzyme in different buffer systems were also considered. This information was computerized, and the rate of glucose phosphorylation in the presence of the mentioned compounds was determined. The value obtained, 1.94 +/- 0.02 mumol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92 +/- 0.3 mumol/h per ml red blood cells). These results provide further evidence for the central role of hexokinase in the regulation of red blood cell glycolysis.

2,3-Diphosphoglycerate↗

PubMed1989

Thioacetamide-induced changes in hepatic hexokinase isoenzymes.

In the 48 h following a single i.p. injection of 200 mg of thioacetamide/kg body wt there is a progressive rise in low-Km hexokinase activity and a concomitant decrease in high-Km glucokinase activity. Loss of glucokinase activity occurs as a consequence of its predominantly perivenous zonal distribution in normal liver since it is hepatocytes in this region of the acinus that are selectively damaged by thioacetamide. Increased low-Km activity is mainly present in inflammatory infiltrates which invade perivenous and mid-zone regions in response to tissue injury. Low- and high-Km hexokinase activities return to control levels 10 days after thioacetamide treatment when the infiltration zones disappear and necrotic perivenous hepatocytes are replaced by cells with normal morphology. Changes in low- and high-Km hexokinase activities induced by thioacetamide thus appear to primarily reflect alterations in cell population rather than changes in gene expression within existing hepatocytes.

Acetamides↗

PubMed1994

Steady state transcript levels of the type II hexokinase and type 1 glucose transporter in human tumor cell lines.

The steady state transcript levels of two hexokinase isozymes and type 1 glucose transporter in human tumor cell lines were analyzed. In HepG2 cells, both type II hexokinase and type 1 glucose transporter were highly expressed. However, in cell lines A431 and HeLa, in which the expression level of type 1 glucose transporter was lower than that in HepG2 cells, the amount of type II hexokinase transcript was almost negligible.

Amino Acid Sequence↗

PubMed1985

Differential effects of monovalent, divalent and trivalent metal ions on rat brain hexokinase.

The effects of monovalent (Li+, Cs+) divalent (Cu2+, Ca2+, Sr2+, Ba2+, Zn2+, Cd2+, Hg2+, Pb2+, Mn2+, Fe2+, Co2+, Ni2+) and trivalent (Cr3+, Fe3+, Al3+) metals ions on hexokinase activity in rat brain cytosol were compared at 500 microM. The rank order of their potency as inhibitors of brain hexokinase was: Cr3+ (IC50 = 1.3 microM) greater than Hg2+ = Al3+ greater than Cu2+ greater than Pb2+ (IC50 = 80 microM) greater than Fe3+ (IC50 = 250 microM) greater than Cd2+ (IC50 = 540 microM) greater than Zn2+ (IC50 = 560 microM). However, at 500 microM Co2+ slightly stimulated brain hexokinase whereas the other metal ions were without effect. That inhibition of brain glucose metabolism may be an important mechanism in the neurotoxicity of metals is suggested.

Animals↗