Heart hexokinase: quaternary structure changes accompanying the binding of regulatory molecules.
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Heart hexokinase monomer has a molecular weight of 97000 and so20,w 5.2 S. It exists in equilibrium with dimer of 194000 molecular weight and so20,w 8.1 S. The proportions of monomer and dimer presence of added ligands are 91% and 9% respectively. The existence of these forms may be demonstrated by separation on electrophoresis or chromatography. In the presence of the regulatory molecule glucose 6-phosphate, the dimer form of the enzyme is favoured. The glucose 6-phosphate mediated dimerisation is abolished in the presence of phosphate or ATP-Mg and less effectively by free ATP. Glucose has no effect on the manomer-dimer equilibrium. On prolonged storage of hexokinase in glucose 6-phosphate polymers are also formed and polymerisation is further enhanced by removal of the ligand. (+info)
Effect of dexamethasone on insulin binding, glucose transport, and glucose oxidation of isolated rat adipocytes.
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We have studied the in vitro effects of dexamethasone on isolated rat adipocytes at concentrations of dexamethasone therapeutically achieved in man. Glucose oxidation, glucose transport, and insulin binding were assessed. In dexamethasone-treated cells, glucose oxidation was decreased by 30-40% both in the absence of insulin (basal state) and at low insulin levels (less than 25 mu/ML). At maximally effective insulin levels (over 100 muU/ml) no differences existed between control and treated cells. If glucose transport were the rate-limiting step for glucose oxidation in the basal state and at low (submaximal) insulin levels, but not at maximally effective insulin concentrations, then these data could be explained by postulating that dexamethasone has a direct affect on glucose transport and does not affect intracellular oxidative pathways. We tested this hypothesis by directly assessing glucose transport in dexamethasone-treated cells. Glucose transport was assessed by measuring the uptake of [14C]2-deoxy glucose. These studies demonstrated a 30-40% decrease in 2-deoxy glucose uptake by treated cells both in the basal state and at all insulin concentrations. Thus, a direct glucocorticoid effect on the glucose transport system seems to account for the decreased ability of dexamethasone-treated cells to oxidize glucose. Since dexamethasone treatment leads to decreased insulin binding to adipocytes in vivo, we examined the possibility that the in vitro decreases in insulin-mediated glucose transport could be due to decreased insulin receptors. Insulin binding to control and treated adipocytes was measured, and no differences were found. Therefore, in cntrast to previously reported in vivo studies, adipocytes treated in vitro with dexamethasone retain a normal ability to bind insulin. Thus, these studies suggest that all of the in vitro effects of dexamethasone on glucose oxidation are due to direct inhibition of the glucose transport system. (+info)
Effect of trimethylcolchicinic acid on the synthesis and excretion of proteoglycans in tissue culture.
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The action of trimethylcolchicinic acid on the synthesis and excretion of proteoglycans has been studied on the L cell strain. The incorporation of precursors has been measured, and proteoglycans produced in the culture medium have been extracted and their concentration determined. The mucopolysaccharide components have been studied by electrophoresis. Control cultures produce hyaluronic acid, dermatan sulfate and very low concentrations of chondroitin 4-sulphate or 6-sulphate. Cultures treated with trimethycolchicinic acid (4 mu g/ml) produce hyaluronic acid, very high concentrations of chondroitin 4-sulphate or 6-sulphate and only traces of dermatan sulphate. So, trimethylcolchicinic acid does not modify the synthesis of hyaluronic acid: it considerably increases the production of chondroitin 4-sulphate or 6-sulphate and inhibits the production of dermatan sulphate. Protein fraction of the proteoglycans is proportionally increased in treated cultures, but there is no marked difference between amino acid concentrations of proteoglycans extracted from control and treated cultures. A slight fall in the cystine concentrations was the only change in the amino acid content of proteoglycans extracted from treated cultures. A hypothesis to explain these results is discussed. (+info)
Intracellular glucose 1-phosphate and glucose 6-phosphate levels modulate Ca2+ homeostasis in Saccharomyces cerevisiae.
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The enzyme phosphoglucomutase plays a key role in cellular metabolism by virtue of its ability to interconvert Glc-1-P and Glc-6-P. It was recently shown that a yeast strain lacking the major isoform of phosphoglucomutase (pgm2Delta) accumulates a high level of Glc-1-P and exhibits several phenotypes related to altered Ca(2+) homeostasis when d-galactose is utilized as the carbon source (Fu, L., Miseta, A., Hunton, D., Marchase, R. B., and Bedwell, D. M. (2000) J. Biol. Chem. 275, 5431-5440). These phenotypes include increased Ca(2+) uptake and accumulation and sensitivity to high environmental Ca(2+) levels. In the present study, we overproduced the enzyme UDP-Glc pyrophosphorylase to test whether the overproduction of a downstream metabolite produced from Glc-1-P can also mediate changes in Ca(2+) homeostasis. We found that overproduction of UDP-Glc did not cause any alterations in Ca(2+) uptake or accumulation. We also examined whether Glc-6-P can influence cellular Ca(2+) homeostasis. A yeast strain lacking the beta-subunit of phosphofructokinase (pfk2Delta) accumulates a high level of Glc-6-P (Huang, D., Wilson, W. A., and Roach, P. J. (1997) J. Biol. Chem. 272, 22495-22501). We found that this increase in Glc-6-P led to a 1.5-2-fold increase in total cellular Ca(2+). We also found that the pgm2Delta/pfk2Delta strain, which accumulated high levels of both Glc-6-P and Glc-1-P, no longer exhibited the Ca(2+)-related phenotypes associated with high Glc-1-P levels in the pgm2Delta mutant. These results provide strong evidence that cellular Ca(2+) homeostasis is coupled to the relative levels of Glc-6-P and Glc-1-P in yeast. (+info)
The pentacovalent phosphorus intermediate of a phosphoryl transfer reaction.
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Enzymes provide enormous rate enhancements, unmatched by any other type of catalyst. The stabilization of high-energy states along the reaction coordinate is the crux of the catalytic power of enzymes. We report the atomic-resolution structure of a high-energy reaction intermediate stabilized in the active site of an enzyme. Crystallization of phosphorylated beta-phosphoglucomutase in the presence of the Mg(II) cofactor and either of the substrates glucose 1-phosphate or glucose 6-phosphate produced crystals of the enzyme-Mg(II)-glucose 1,6-(bis)phosphate complex, which diffracted x-rays to 1.2 and 1.4 angstroms, respectively. The structure reveals a stabilized pentacovalent phosphorane formed in the phosphoryl transfer from the C(1)O of glucose 1,6-(bis)phosphate to the nucleophilic Asp8 carboxylate. (+info)
Glycogen synthesis in the perfused liver of streptozotocin-diabetic rats.
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1. Net glycogen accumulation was measured in sequentially removed samples during perfusion of the liver of starved streptozotocin-diabetic rats, and shown to be significantly impaired, compared with rates in normal (starved) rats. 2. In perfusions of normal livers with glucose plus C3 substrates, there was an increase in the proportion of glycogen synthetase 'a', compared with that in the absence of substrates. This response to substrates, followed in sequential synthesis and enzymic sensitivity in the perfused liver of diabetic rats were reversed by pretreatment in vivo with glucose plus fructose, or insulin. Glucose alone did not produce this effect. 4. Glucose, fructose, insulin or cortisol added to e perfusion medium (in the absence of pretreatment in vivo) did not stimulate glycogen synthesis in diabetic rats. 5. In intact diabetic rats, there was a decline in rates of net hepatic glycogen accumulation, and the response of glycogen synthetase to substrates. The most rapid rates of synthesis were obtained after fructose administration. 6. These results demonstrate that there is a marked inherent impairment in hepatic glycogen synthesis in starved diabetic rats, which can be rapidly reversed in vivo but no in perfusion. Thus hepatic glycogen synthesis does not appear to be sensitive to either the short-term direct action of insulin (added alone to perfusions) of to long-term insulin deprivation in vivo. The regulatory roles of substrates, insulin and glycogen synthetase in hepatic glycogen accumulation are discussed. (+info)
Functional insights revealed by the crystal structures of Escherichia coli glucose-1-phosphatase.
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The Escherichia coli periplasmic glucose-1-phosphatase is a member of the histidine acid phosphatase family and acts primarily as a glucose scavenger. Previous substrate profiling studies have demonstrated some of the intriguing properties of the enzyme, including its unique and highly selective inositol phosphatase activity. The enzyme is also potentially involved in pathogenic inositol phosphate signal transduction pathways via type III secretion into the host cell. We have determined the crystal structure of E. coli glucose-1-phosphatase in an effort to unveil the structural mechanism underlying such unique substrate specificity. The structure was determined by the method of multiwavelength anomalous dispersion using a tungstate derivative together with the H18A inactive mutant complex structure with glucose 1-phosphate at 2.4-A resolution. In the active site of glucose-1-phosphatase, there are two unique gating residues, Glu-196 and Leu-24, in addition to the conserved features of histidine acid phosphatases. Together they create steric and electrostatic constraints responsible for the unique selectivity of the enzyme toward phytate and glucose-1-phosphate as well as its unusually high pH optimum for the latter. Based on the structural characterization, we were able to derive simple structural principles that not only precisely explains the substrate specificity of glucose-1-phosphatase and the hydrolysis products of various inositol phosphate substrates but also rationalizes similar general characteristics across the histidine acid phosphatase family. (+info)
Investigation of ion/molecule reactions as a quantification method for phosphorylated positional isomers. an FT-ICR approach.
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A rapid and accurate method of quantifying positional isomeric mixtures of phosphorylated hexose and N-acetylhexosamine monosacchrides by using gas-phase ion/molecule reactions coupled with FT-ICR mass spectrometry is described. Trimethyl borate, the reagent gas, reacts readily with the singly charged negative ions of phosphorylated monosaccharides to form two stable product ions corresponding to the loss of one or two neutral molecules of methanol from the original adduct. Product distribution in the ion/molecule reaction spectra differs significantly for isomers phosphorylated in either the 1- or the 6-position. As a result, the percents of total ion current of these product ions for a mixture of the two isomers vary with its composition. In order to determine the percentage of each isomer in an unknown mixture, a multicomponent quantification method is utilized in which the percents of total ion current of the two product ions for each pure monosaccharide phosphate and the mixture are used in a two-equation, two-unknown system. The applicability of this method is demonstrated by successfully quantifying mock mixtures of four different isomeric pairs: Glucose-1-phosphate and glucose-6-phosphate; mannose-1-phosphate and mannose-6-phosphate; galactose-1-phosphate and galactose-6-phosphate; N-acetylglucosamine-1-phosphate and N-acetylglucosamine-6-phosphate. The effects of mixture concentrations and ion/molecule reaction conditions on the quantification are also discussed. Our results demonstrate that this assay is a fast, sensitive, and robust method to quantify isomeric mixtures of phosphorylated monosaccharides. (+info)