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PubMedDate not supplied

[Activity, hexokinase isoenzyme spectrum and various factors of their regulation in liver and skeletal muscles of young piglets].

Hexokinase in the liver of 1- and 5-day-old piglets is presented by four isoforms and in the skeletal muscles--by two ones. The enzyme activity in the liver and skeletal muscles of 5-day-old piglets is much higher than in 1-day-old ones. The increased hexokinase activity in the tissues of piglets during the first days of life appears to be due to the changes in their isoenzyme spectrum. The hexokinase activity and isoenzyme spectrum in the investigated tissue were affected by insulin, cortisol and 24 hours long starvation. These changes depended upon the age of the animals and differed in various organs and tissues: in 1-day-old piglets they were more pronounced in the skeletal muscles, while in 5-days-old animals--in the liver.

Aging↗

PubMedDate not supplied

[Possible significance of hexokinase binding to mitochondrial membranes].

A magnitude of the Pasteur's effect and content of bound hexokinase were studied in various malignant and normal tissues. In all cases studied these two values were practically the same. Dynamics of changes in subcellular localization of hexokinase and the Pasteur's effect were similarly altered in liver tissue during ontogenesis. Binding hexokinase with mitochondrial membranes was assumed to be the cytophysiological mechanism for regulation of glycolytic activity by respiration.

Animals↗

PubMed1991

Glucose phosphorylation. Site-directed mutations which impair the catalytic function of hexokinase.

Recent studies from this and other laboratories have resulted in the cloning and sequencing of hexokinases from a variety of tissues including yeast, human kidney, rat brain, rat liver, and mouse hepatoma. Significantly, studies on the hepatoma enzyme conducted in this laboratory (Arora, K.K., Fanciulli, M., and Pedersen, P.L. (1990) J. Biol. Chem. 265, 6481-6488) resulted also in its overexpression in Escherichia coli in active form. We have now used site-directed mutagenesis for the first time in studies of hexokinase to evaluate the role of amino acid residues predicted to interact with either glucose or ATP. Four amino acid residues (Ser-603, Asp-657, Glu-708, and Glu-742) believed to interact with glucose were mutated to alanine or glycine, whereas a lysine residue (Lys-558) thought to be directly involved in binding ATP was mutated to either methionine or arginine. Of all the mutations in residues believed to interact with glucose, the Asp-657----Ala mutation is the most profound, reducing the hexokinase activity to a level less than 1% of the wild type. The relative Vmax values for Ser-603----Ala, Glu-708----Ala, and Glu-742----Ala enzymes are 6, 10, and 6.5%, respectively, of the wild-type enzyme. Glu-708 and Glu-742 mutations increase the apparent Km for glucose 50- and 14-fold, respectively, while the Ser-603----Ala mutation decreases the apparent Km for glucose 5-fold. At the putative ATP binding site, the relative Vmax for Lys-558----Arg and Lys-558----Met enzymes are 70 and 29%, respectively, of the wild-type enzyme with no changes in the apparent Km for glucose. No changes were observed in the apparent Km for ATP with any mutation. These results support the view that all 4 residues predicted to interact with glucose from earlier x-ray studies may play a role in binding and/or catalysis. The Asp-657 and Ser-603 residues may be involved in both, while Glu-708 and Glu-742 clearly contribute to binding but are not essential for catalysis. In contrast, Lys-558 appears to be essential neither for binding nor catalysis.

Amino Acids↗

PubMed1990

Effect of experimental diabetes on the activity of hexokinase isoenzymes in tissues of the rat.

The effect of experimental diabetes on the activity of hexokinase isoenzymes was studied in a wide range of tissues of the rat. In the tissues known to require insulin for glucose phosphorylation, the activity of hexokinase was markedly decreased; the fall being mainly in the Type IV (Glucokinase) in liver and Type II in other tissues, these tissues also exhibit glucose underutilization in diabetes. In the tissues which are commonly known not to require insulin, the activity of Type I hexokinase was significantly increased, these tissues exhibit aspects of glucose overutilization in diabetes in particular kidney and lens. These changes are discussed in relation to Spiro's hypothesis of glucose under and overutilization in tissues in diabetes.

Animals↗

PubMed1990

[Adsorption mechanism of hexokinase activity regulation in rat sarcoma M-1].

The hexokinase interaction with mitochondrial membranes in rat sarcoma M-1 cells was studied. The conditions of formation of the enzyme complex with mitochondrial membranes and its stability were elaborated. The kinetic parameters of free and membrane-bound hexokinases were determined. The data obtained are discussed within the frame-work of the adsorption mechanism of hexokinase activity regulation in neoplastic tissues.

Adsorption↗

PubMedDate not supplied

[Effect of histamine on the hexokinase activity and RNA synthesis in the seminal vesicles and prostate of orchiectomized rats].

Due to orchidectomy, carried out two weeks before the experiment, in androgene-dependent rat tissues a hexokinase activity was found to be decreased several times. Within 24 hrs after administration of histamine into orchidectomized rats the hexokinase activity was increased by 96% in seminal follicles and by 160% in prostate. Incorporation of 3H-uridine into RNA of seminal follicles and prostate of castrated rat males was increased by 60% and 210%, respectively, within 3 hrs after the administration of histamine. The stimulating effects of histamine on RNA synthesis and the hexokinase activity were completely inhibited by actinomycin D. The data obtained suggest that histamine was one of mediators in the effect of testosterone on the tissues-targets.

Age Factors↗

PubMed1989

Role of hexokinase in controlling the glucose metabolism flux: a study of its flux control coefficient in different tissues.

Flux control coefficients of hexokinase for glucose metabolism in different rat tissues have been determined, showing that the hepatocyte cytosolic hexokinase is the only one which plays an important role in the control of the glucose-input flux studied among the different tissues. Explanation of these results are given in terms of the kinetics features of hexokinase and the metabolic role of glucose in these tissues.

Animals↗

PubMed1989

Kinetic properties of hexokinase under near-physiological conditions. Relation to metabolic arrest in Artemia embryos during anoxia.

Previous analyses of glycolytic metabolites in Artemia embryos indicate that an acute inhibition of glucose phosphorylation occurs during pHi-mediated metabolic arrest under anoxia. We describe here kinetic features of hexokinase purified from brine shrimp embryos in an attempt to explain the molecular basis for this inhibition. At saturating concentrations of cosubstrate, ADP is an uncompetitive inhibitor toward glucose and a partial noncompetitive inhibitor toward ATP (Kis = 0.86 mM, Kii = 1.0 mM, Kid = 1.9 mM). With cosubstrates at subsaturating concentrations, the uncompetitive inhibition versus glucose becomes noncompetitive, while inhibition versus ATP remains partial noncompetitive. The partial noncompetitive inhibition of ADP versus ATP is characterized by a hyperbolic intercept replot. These product inhibition patterns are consistent with a random mechanism of enzyme action that follows the preferred order of glucose binding first and glucose-6-P dissociating last. We propose that inhibition by glucose-6-P (Kis = 65 microM) occurs primarily by competing with ATP at the active site, resulting in the formation of the dead-end complex, enzyme-glucose-glucose-6-P. Versus glucose, inhibition by glucose-6-P is uncompetitive at pH 8.0 and noncompetitive at pH 6.8. Over a physiologically relevant pH range of 8.0 to 6.8 alterations in Km and Ki values do not account for the reduction in glucose phosphorylation, and no evidence suggests that Artemia hexokinase activity is modulated by reversible binding to intracellular structures. Total aluminum in the embryos is 4.01 +/- 0.36 micrograms/g dry weight, or, based upon tissue hydration, 72 microM. This concentration of aluminum dramatically reduces enzyme activity at pH values less than 7.2, even in the presence of physiological metal ion chelators (citrate, phosphate). When pH, aluminum, citrate, phosphate, substrates, and products were maintained at cellular levels measured under anoxia, we can account for a 90% inhibition of hexokinase relative to activity under control (aerobic) conditions.

Aerobiosis↗

PubMed1986

[The role of adenylate kinase in the regulation of the rate and effectiveness of energy transfer from mitochondria to hexokinase in vitro].

The effect of adenylate kinase activity on the rate and efficiency of energy transport from mitochondria to hexokinase was studied in a system containing isolated rabbit heart mitochondria, hexokinase and adenylate kinase at low concentrations of adenine nucleotides. Oxygen consumption by mitochondria and glucose-6-phosphate synthesis by hexokinase were recorded. It was found that with adenylate kinase being active both in mitochondria and in the washing solution, the rate and efficiency of glucose-6-phosphate synthesis considerably increases. The effects of adenylate kinase activity are fully abolished by diadenosine pentaphosphate, an inhibitor of adenylate kinase. The experimental results based on the use of adenylate kinase demonstrate the possibility of increasing the rate and efficiency of energy transfer between two spatially uncoupled biochemical processes in vitro with the aid of an enzymatic system.

Adenylate Kinase↗

PubMed1988

The adenine nucleotide binding site on yeast hexokinase PII. Affinity labeling of Lys-111 by pyridoxal 5'-diphospho-5'-adenosine.

The adenine nucleotide analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), is shown to be a potent and specific inhibitor of yeast hexokinase PII. Evidence that the analog binds specifically at the ATP binding site includes the demonstration that glucose binding enhances PLP-AMP binding and that PLP-AMP and ATP bind competitively with an apparent Ki(PLP-AMP) = 23 microM. In addition, from the relationship between the degree of inhibition and extent of modification, it is estimated that the incorporation of 1 mol of PLP-AMP/mol of subunit is required for complete inhibition. Borohydride reduction of the Schiff's base complex formed between hexokinase and [3H]PLP-AMP gives a stable product. The reduced derivative was digested with trypsin and a single radioactive peptide was isolated by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis identified Lys-111 as the modified residue. Taking into account the known structures of the binary complexes (Shoham, M., and Steitz, T. A. (1980) J. Mol. Biol. 140, 1-14), the results suggest that Lys-111, located in the smaller of the two lobes of hexokinase, moves into the active site upon formation of the ternary complex.

Adenine Nucleotides↗

PubMed1977

Yeast hexokinase mutants.

Using yeast mutants, it is shown that growth on glucose occurs even in the absence of both hexokinase A and hexokinase B; fructose growth requires at least one of these two enzymes. Expression of hexokinase A and of glucokinase seem to be regulated.

Culture Media↗

PubMed1986

[Isolation of hexokinase II isoenzyme from the hyaloplasm of rat skeletal muscle].

A method for obtaining partially purified preparation of II isozyme of hexokinase from cytosol of the rat skeletal muscles has been suggested. According to the data of electrophoretic and kinetic analyses the preparation does not practically contain I isozyme of hexokinase and is characterized by high enzymatic activity. The obtained preparation of II isozyme of hexokinase may be successfully used for research of the adsorption mechanism controlling the enzyme activity.

Animals↗

PubMedDate not supplied

Role of hexokinase in the regulation of glucose metabolism in human erythrocytes.

Red blood cell glucose metabolism was studied in erythrocytes from a patient with trisomy 10 p which resulted in + 50% hexokinase specific activity, in normal controls and in cases of heterozygous hexokinase deficiency. The results obtained show that the hexokinase activity level is an important factor in the control of the erythrocyte's glycolytic rate while having no appreciable effect on the hexose monophosphate pathway under resting conditions. No clear conclusion could be drawn when an oxidative stress was present.

Blood Glucose↗

PubMed1985

Anomeric specificity of hexokinase in rat, human, and murine tumor cells.

In tumoral cells derived from the insulin-producing rat cell line RINm5F, both low- and high-Km glucose-phosphorylating enzymic activities were present. The hexokinase-like enzyme was inhibited by glucose 6-phosphate and displayed a greater affinity for but lower maximal velocity with alpha-D-glucose than beta-D-glucose. A comparable anomeric behavior of hexokinase was observed in breast cancer (MCF-7) and lymphocytic leukemia (P388) cells. Thus, the anomeric specificity of hexokinase in tumoral cells was not different from that recently characterized in normal mammalian cells.

Animals↗

PubMed1985

Direct spectrophotometry of magnesium in serum after reaction with hexokinase and glucose-6-phosphate dehydrogenase.

We describe a simple method for determining magnesium in serum by using hexokinase (EC 2.7.1.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The method is based on determination of the reaction rate of hexokinase activated by Mg2+, which participates in the hexokinase reaction as the substrate in the form of a Mg X ATP2- complex. The reaction rate is determined from the change in absorbance at 340 nm as NADPH is produced by the action of glucose-6-phosphate dehydrogenase. This simple and rapid spectrophotometric method does not require expensive instrumentation, but results correlate satisfactorily with those obtained by atomic absorption spectroscopy. Thus, the present method gives a "true" value for magnesium in serum, a value appreciably lower than that obtained by an earlier colorimetric method, the Xylidyl Blue II method (Biochem Med 7: 208-217, 1973), which lacks specificity.

Adenosine Triphosphate↗

PubMedDate not supplied

[Changes in the activity and isoenzymic spectrum of skeletal muscle and brain hexokinase under organism adaptation to intensive physical exercise].

The activity of hexokinase and its isoenzymic spectrum were determined in skeletal muscles of different metabolic types and in brain of trained and untrained albino rats. It is shown that adaptation to intensive muscular activity accompanied by metabolic acidosis induces some changes in the hexokinase system, which consist in the increase of the total hexokinase activity not only under the optimal conditions but also with more acidic pH values of the medium and in the rise of the quantity of the muscular type isoenzyme which is more stable to the pH action.

Acidosis↗

PubMedDate not supplied

Rat lung hexokinase: some aspects of the metabolic and hormonal regulation of its subcellular distribution.

Addition of glucose-6-phosphate (0.03-0.06 mM) to a suspension of rat lung mitochondria promotes solubilization of 30 to 85% of the bound form of hexokinase. This effect is significantly enhanced by the simultaneous addition of glucose (10 mM), fructose (10 mM) or mannose (10 mM). Conversely addition of N-acetylglucosamine (10 mM), mannose-6-phosphate (10 mM) or inorganic phosphate (1 mM) reduces hexokinase solubilization. Insulin is involved in the regulation of the interaction between mitochondrial membrane and hexokinase. Treatment with alloxan causes an increase of the free form of the enzyme in the lung and a correspondent reduction of the bound form. Insulin administration restores the normal intracellular distribution of the enzyme.

Animals↗

PubMedDate not supplied

Purification and properties of the cytoplasmic hexokinase from rabbit brain.

About 90% of the total hexokinase activity in rabbit brain was found to be associated with mitochondria while the remaining part was found in the cytosolic fraction. The soluble enzyme was purified 4,700-fold to near homogeneity by a combination of ion-exchange chromatography, dye-ligand chromatography and affinity chromatography. The purified enzyme showed a specific activity of 110 units/mg of protein and was obtained in 70% yield. The molecular weight of the purified hexokinase was found to be approximately 98,000 both for the native and the denatured enzyme. The isoelectric point, pI, was 6.2 pH units by isoelectric focusing and the enzyme was found to be able to phosphorylate several hexoses. Mg . ATP2-, among the nucleotide substrates, was the most effective phosphate donor. The properties of the purified cytoplasmatic hexokinase were compared with those of the solubilized mitochondrial enzyme. No significant differences were found in molecular weight, isoelectric point, pH dependence of activity, electrophoretic mobility and affinity for glucose and Mg.ATP2-. However, the temperature dependence of activity, and the specificity for several hexose substrates were markedly different.

Animals↗