Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.1 (hexokinase)
 5,274 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Individual hexokinase isoenzymes (isoHK) are isolated from normal and malignant human stomach mucosa. IsoHK from tumour tissue are found to have KM for glucose 10 times as low as isoHK from normal tissue. Molecular weights of individual isoHK from normal and tumour tissues are similar (at the range of 112,000-125,000). The treatment of protein preparation with 8M urea in the presence of 1% sodium docecyl sulphate resulted in the appearance of a single band with molecular weight of 58,000-60,000 for all the isoHK under polyacrylamide gel electrophoresis. Intensive bands with molecular weight of 60,000 and 96,000 and a number of minor bands were observed under polyacrylamide gel disc elect-ophoresis in the absence of urea. 2-Mercaptoethanol did not affect the results of disc electrophoresis. It is concluded that the molecule of human hexokinase consists of two subunits with molecular weight of 60,000.
 ...
PMID:[Physico-chemical and kinetic properties of hexokinase isoenzymes from human normal and tumor tissues]. 121 49

Yeast hexokinase is a homodimer consisting of two identical subunits. Yeast hexokinase was inactivated by 2-aminothiophenol at 25 degrees C (pH 9.1). The reaction followed pseudo-first-order kinetics until about 70% of the phosphotransferase activity was lost. About 0.65 mol of 2-aminothiophenol/mol of hexokinase was found to be bound after the 70% loss of the enzyme activity. Completely inactivated hexokinase showed a stoichiometry of about 1 mol of 2-aminothiophenol bound/mol of the enzyme. The evidence obtained from kinetic experiments, stoichiometry of the inactivation reaction and fluorescence emission measurements suggested site-site interaction (weak negative co-operativity) during the inactivation reaction. The approximate rate constants for the reversible binding of 2-aminothiophenol to the first subunit (KI) and for the rate of covalent bond formation with only one site occupied (k3) were 150 microM and 0.046 min-1 respectively. The inactivation reaction was pH-dependent. Dithiothreitol, 2-mercaptoethanol and cysteine restored the phosphotransferase activity of the hexokinase after inactivation by 2-aminothiophenol. Sugar substrates protected the enzyme from inactivation more than did the nucleotides. Thus it is concluded that the inactivation of the hexokinase by 2-aminothiophenol was a consequence of a covalent disulphide bond formation between the aminothiol and thiol function at or near the active site of the enzyme. Hexokinase that had been completely inactivated by 2-aminothiophenol reacted with o-phthalaldehyde. Fluorescence emission intensity of the incubation mixture containing 2-aminothiophenol-modified hexokinase and o-phthalaldehyde was one-half of that obtained from an incubation mixture containing hexokinase and o-phthalaldehyde under similar experimental conditions. The intensity and position of the fluorescence emission maximum of the 2-aminothiophenol-modified hexokinase were different from those of the native enzyme, indicating conformational change following modification. Whereas aliphatic aminothiols were completely ineffective, aromatic aminothiols were good inhibitors of the hexokinase. Cyclohexyl mercaptan weakly inhibited the enzyme. Inhibition of the hexokinase by heteroaromatic thiols was dependent on the nature of the heterocyclic ring and position of the thiol-thione equilibrium. The inhibitory function of a thiol is associated with the following structural characteristics: (a) the presence of an aromatic ring, (b) the presence of a free thiol function and (c) the presence of a free amino function in the close proximity of the thiol function.(ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:Inactivation of yeast hexokinase by 2-aminothiophenol. Evidence for a 'half-of-the-sites' mechanism. 284 99

Skeletal muscle actin labelled with pyrene was used to measure the critical concentration (Cc) for assembly in conditions designed to approximate the ionic environment in the cytoplasm. Under these conditions (0.1 M-KCl, 2 mM-MgCl2, 1.1 mM-ATP, 0.1 mM-CaCl2, 0.5 mM-ethyleneglycol-bis(beta-aminoethylether)N,N'-tetraacetic acid, 0.25 mM-2-mercaptoethanol, 20 mM-imidazol X HCl, pH 7.0), the steady-state Cc value was estimated to be 0.07 microM (3.0 micrograms/ml), and, consistent with previous observations, the Cc increased to 0.20 microM (8.7 micrograms/ml) in the presence of 10(-6) M-cytochalasin D, and to 1.10 microM (47 micrograms/ml) after conversion of ATP to ADP using hexokinase and glucose. Addition of inorganic phosphate (Pi) at concentrations up to 20 mM caused only a slight decrease in the steady-state Cc, but at 2 mM-Pi (a reasonable estimate of cytoplasmic concentrations) the increase in Cc due to cytochalasin D was abolished, and at higher Pi concentrations there was even a slight decrease. Increasing Pi concentrations also progressively reduced the steady-state Cc for ADP-actin close to that for ATP-actin. These results are consistent with an increased affinity of ADP-actin for the polymer in the presence of Pi. To determine whether these effects of Pi were simply mass action effects on hydrolysis of bound ATP by polymerized actin, the stoichiometry of ATP hydrolysis during actin assembly was estimated and found to be at unity within the limits of experimental error and to be unaffected by Pi up to 20 mM. In addition, actin depolymerized by removal of ATP using glucose and hexokinase rapidly reassembled after addition of 20 mM-Pi. These results are interpreted by a mechanism involving the formation of ADP-Pi-actin species and are discussed in relation to the phenomenon of treadmilling and the theory of dynamic instability, and the potential for their occurrence in cells.
 ...
PMID:Cytoplasmic concentrations of inorganic phosphate affect the critical concentration for assembly of actin in the presence of cytochalasin D or ADP. 380 73

M. Kuwajima, C. B. Newgard, D. W. Foster, and J. D. McGarry (1986, J. Biol. Chem. 261, 8849-8853) have concluded that the reason postprandial hepatic glycogenesis occurs primarily from gluconeogenic precursors rather than glucose is because glucokinase activity is insufficient to support the observed rates of glycogen synthesis. F. L. Alvares and R. C. Nordlie (1977, J. Biol. Chem. 252, 8404-8414) have concluded that the combined activities of glucokinase and hexokinase are less than the apparent rates of hepatic glucose uptake. We have identified several factors in the assays used in these studies which lead to substantial underestimations of glucokinase activity. Glucokinase was assayed either by allowing glucose 6-phosphate to accumulate over 10 min (discontinuous assay) or by coupling the formation of glucose 6-phosphate with its oxidation by Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase and NAD (continuous assay). Accurate determinations of glucokinase at 37 degrees C with subsaturating glucose require both 100 mM KCl and 2.5 mM dithioerythritol in the assay medium; 2-mercaptoethanol will not substitute for dithioerythritol. When both KCl and dithioerythritol are absent (Kuwajima et al.) glucokinase activity is underestimated by 3- to 5-fold. The discontinuous assay as used previously (Alvares and Nordlie) underestimates glucokinase activity in crude extracts by 2- to 2.5-fold, due in part to the hydrolysis of glucose 6-phosphate and its transformation to other hexose monophosphates. Under optimized conditions at 37 degrees C both assays yield similar results in extracts from fed rats, i.e., 2-3 and 4-5 units/g liver at 10 and 100 mM glucose, respectively. Some implications of the finding that total hepatic glucose phosphorylating capacity at physiological concentrations significantly exceeds the observed rates of postprandial glycogen synthesis are discussed.
 ...
PMID:Factors underlying significant underestimations of glucokinase activity in crude liver extracts: physiological implications of higher cellular activity. 381 60