Detection of fructose-1,6-diphosphatase deficiency with use of white blood cells.
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Adult↗
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Sounding Board. A possible metabolic basis for the control of body weight.
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Adenosine Diphosphate↗
The acute biochemical response of the starved rabbit liver in situ to glucose infusion.
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Adenosine Monophosphate↗
Triosephosphate metabolism by mature boar spermatozoa.
Boar sperm rapidly interconverted dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, produced fructose-1,6-bisphosphate, approximately equilibrium concentrations of fructose 6-phosphate and glucose 6-phosphate but not glycerol or glycerol 3-phosphate. In the presence of 3-chloro-1-hydroxypropanone, an inhibitor of stage 2 of the glycolytic pathway, the triosephosphates were metabolized faster, produced less fructose-1,6-bisphosphate, fructose 6-phosphate and glucose 6-phosphate, but not glycerol or glycerol 3-phosphate. This suggests that these cells may have the capacity to convert glycolytic intermediates into a storage metabolite to conserve carbon atoms for the eventual synthesis of lactate.
Acetone↗
Fructose metabolism by mature boar spermatozoa.
In 1945, Mann showed fructose to be the principal sugar in semen. For over half a century the means by which fructose is metabolized by sperm has been assumed to be by an initial phosphorylation catalysed by hexokinase, but this has never been substantiated. In the present study, by comparing the metabolism of glucose and fructose by both whole boar sperm and hypotonically treated cells, it is confirmed that fructose is phosphorylated by hexokinase to produce fructose 6-phosphate.
Animals↗
Metabolism of fructose-1,6-bisphosphate by mature boar spermatozoa.
Mature epididymal boar spermatozoa converted glucose and fructose to carbon dioxide and lactate and maintained high concentrations of ATP. In the presence of (S)-alpha-chlorohydrin these processes were inhibited and there was an accumulation of fructose-1,6-bisphosphate and dihydroxyacetone phosphate. With fructose-1,6-bisphosphate as the substrate, the concentration of ATP was maintained, carbon dioxide was evolved and dihydroxyacetone phosphate accumulated. Cells pre-incubated with (S)-alpha-chlorohydrin did not maintain ATP levels, evolved less carbon dioxide and produced dihydroxyacetone phosphate. Assays of incubates in which fructose-1,6-bisphosphate was used as the substrate showed the presence of equilibrium quantities of fructose-6-phosphate and glucose-6-phosphate which were not detected when either fructose or glucose were used as substrates. [14C]Fructose and [14C]glucose were not produced from [14C]fructose-1,6-bisphosphate in spermatozoal incubates which had or had not been pre-incubated with (S)-alpha-chlorohydrin. Evidence is presented that a high concentration of fructose-1,6-bisphosphate leads to the formation of fructose-6-phosphate and glucose-6-phosphate but not of fructose and/or glucose.
Adenosine Diphosphate↗
Purification, molecular cloning, and sequence analysis of sucrose-6F-phosphate phosphohydrolase from plants.
Sucrose-6(F)-phosphate phosphohydrolase (SPP; EC ) catalyzes the final step in the pathway of sucrose biosynthesis and is the only enzyme of photosynthetic carbon assimilation for which the gene has not been identified. The enzyme was purified to homogeneity from rice (Oryza sativa L.) leaves and partially sequenced. The rice leaf enzyme is a dimer with a native molecular mass of 100 kDa and a subunit molecular mass of 50 kDa. The enzyme is highly specific for sucrose 6(F)-phosphate with a K(m) of 65 microM and a specific activity of 1250 micromol min(-1) mg(-1) protein. The activity is dependent on Mg(2+) with a remarkably low K(a) of 8-9 microM and is weakly inhibited by sucrose. Three peptides from cleavage of the purified rice SPP with endoproteinase Lys-C showed similarity to the deduced amino acid sequences of three predicted open reading frames (ORF) in the Arabidopsis thaliana genome and one in the genome of the cyanobacterium Synechocystis sp. PCC6803, as well as cDNA clones from Arabidopsis, maize, and other species in the GenBank database of expressed sequence tags. The putative maize SPP cDNA clone contained an ORF encoding a 420-amino acid polypeptide. Heterologous expression in Escherichia coli showed that this cDNA clone encoded a functional SPP enzyme. The 260-amino acid N-terminal catalytic domain of the maize SPP is homologous to the C-terminal region of sucrose-phosphate synthase. A PSI-BLAST search of the GenBank database indicated that the maize SPP is a member of the haloacid dehalogenase hydrolase/phosphatase superfamily.
Amino Acid Sequence↗
Changes in rabbit-liver lysosomes and fructose 1,6-bisphosphatase induced by cold and fasting.
Exposure of rabbits to cold or fasting results in marked increases in the number and size of liver lysosomes, associated with increases in the total and "free" proteolytic activity. Changes in lysosomal morphology also indicate increased activity and fragility. These effects of cold and fasting are correlated with decreases in the levels of liver fructose bisphosphatase (EC 3.1.3.11) activity and changes in the molecular properties of the purified enzyme, including the loss of a small peptide containing the only tryptophan residue in the peptide chain.
Animals↗
Inactive enzyme molecules in aging mice: liver aldolase.
Evidence is presented that there is a considerable accumulation of inactive fructose-1,6-diphosphate aldolase (EC 4.1.2.7) in the liver of senescent mice. Liver aldolase was purified from 3-month-old mice and used to immunize rabbits. It was demonstrated with the monospecific antibody thus produced that the liver aldolase of young adult (3 month) and aged (31 month) mice are antigenically identical. With the antibody, inactive enzyme molecules (crossreacting material) in liver homogenate of old mice were detected. The liver aldolase of senescent mice had half as much active enzyme per mg of protein, as well as per antigenic unit, as did the liver aldolase of young adult mice. The accumulation of faulty enzyme molecules may be one of the causes of debilitation leading to senescence and death.
Aging↗
Glucose inhibition of epinephrine stimulation of hepatic gluconeogenesis by blockade of the alpha-receptor function.
For isolated rat hepatocytes, glucagon, 3':5'-cyclic AMP, 3':5'-cyclic GMP, and epinephrine stimulate the rate of gluconeogenesis from substrates not involving pathways of mitochondrial metabolism. From estimation of the rates of glucose formation, fructose 6-phosphate phosphorylation, and lactate and pyruvate formation it is concluded that epinephrine and 3':5'-cyclic GMP stimulate gluconeogenesis from either galactose or fructose by influencing the rate of reactions involving fructose 6-phosphate in a manner similar to that already reported for glucagon and 3':5'-cyclic AMP. Each agent acts to inhibit flux through phosphofructokinase (EC 2.7.1.11) and enhance flux through fructose diphosphatase (EC 3.1.3.11), resulting in the re-direction of carbon from lactate and pyruvate formation to glucose synthesis. In addition to 3':5'-cyclic GMP, dibutyryl 3':5'-cyclic GMP, 8-bromo 3':5'-cyclic GMP, 8-benzyl-thio 3':5'-cyclic GMP and 8-(4-chlorophenyl)thio 3':5'-cyclic GMP stimulate glucose formation and inhibit lactate and pyruvate formation from galactose. Guanosine monophosphate and 2':3'-cyclic GMP are inactive. As the stimulatory effect of epinephrine is inhibited by phenoxybenzamine and not by propranolol, and is not simulated by isoproterenol, it is concluded that catecholamine activity is expressed through the alpha-receptor. Increased extracellular glucose concentration (>10 mM) decreases the stimulatory effect of epinephrine, 3':5'-cyclic GMP, and partially that of 3':5'-cyclic AMP but does not alter the efficacy of glucagon.
Adrenergic alpha-Antagonists↗
The explanation for the blockade of glycolysis in early mouse embryos.
The reason for the failure of early-stage mouse embryos to grow on glucose alone was investigated by measurement of glucose-6-phosphate, fructose-1,6-bisphosphate plus triose phosphates, citrate, and malate in individual embryos during starvation and refeeding with glucose or glucose plus pyruvate. The results indicate a block at the 6-phosphofructokinase (EC 2.7.1.11) step at early stages, which is later removed. Although there seems to be no early difficulty in phosphorylation of glucose, maximum glucose-6-phosphate levels (and probably fructose-6-phosphate levels) are much lower at early stages than at later stages. The increase in fructose-6-phosphate with age may be the major cause of the increase in 6-phosphofructokinase activity. Unusually high citrate levels at all ages may help to keep this enzyme strongly inhibited until the increase in fructose-6-phosphate occurs. The changes in metabolite levels also indicate an early defect in mobilization of glycogen and a probably less important defect in the citrate cycle.
Animals↗
31P nuclear magnetic resonance studies of Ehrlich ascites tumor cells.
High-resolution 31P nuclear magnetic resonance spectra at 145.7 MHz are reported for intact Ehrlich ascites tumor cells and their perchloric acid extracts. In the extracts it was possible to assign resonances to fructose 1,6-bisphosphates, dihydroxyacetone phosphate, ATP, ADP, AMP, Pi, NAD+, phosphorylcholine, glycero-3-phosphorylcholine, glycero-3-phosphorylethanolamine, and glyceraldehyde 3-phosphate from their chemical shifts, pH behavior, and spin couplings. All but glyceraldehyde 3-phosphate were observed and assigned in the intact cells. It was possible to show that the hydrolysis of fructose 1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate is in equilibrium, that the dihydroxyacetone phosphate leads to glyceraldehyde 3-phosphate reaction is not, and that in the intact cell without added oxygen or glucose the reaction 2ADP in equilibrium ATP + AMP is in equilibrium. From the known pH dependence of the Pi resonance it was possible to show that during aerobic or anerobic glycolysis the difference between intracellular and extracellular pH values was less than 0.2 pH units. Upon oxygenation the ATP concentration increased while the ADP concentration fell. Introducing deoxyglucose depleted the ATP and resulted in an AMP signal and one from deoxyglucose 6-phosphate, which is transported and phosphorylated but not catabolized.
Adenine Nucleotides↗
Control of liver 6-phosphofructokinase by fructose 2,6-bisphosphate and other effectors.
Rat liver 6-phosphofructokinase (ATP-D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was partially purified free of interfering enzymes by a rapid procedure. Fructose 2,6-bisphosphate, at micromolar concentrations, greatly stimulated the enzyme by increasing its affinity for fructose 6-phosphate and relieving the inhibition by ATP. Its action was synergistic with that of AMP. As a stimulator of liver phosphofructokinase, fructose 2,6-bisphosphate was approximately 1000- and 2500-fold more efficient than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate, respectively. The concentration at which a half-maximal effect was obtained with the hexose bisphosphates was dependent upon the experimental conditions. It was relatively high at physiological concentrations of substrates, AMP, and Pi, and under these conditions the positive effect of fructose 1,6-bisphosphate was no longer detectable. This was probably due to the negative effect of fructose 1,6-bisphosphate as a reaction product inhibitor. It is concluded that fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate controls, in association with other effectors, the activity of phosphofructokinase in the liver.
Adenosine Monophosphate↗
Mannose 6-, fructose 1-, and fructose 6-phosphates inhibit human natural cell-mediated cytotoxicity.
In vitro human natural cell-mediated cytotoxicity (NCMC) to K-562, Molt-4, and F-265 cells is inhibited in a dose-dependent manner by mannose 6-phosphate, fructose 1-phosphate and fructose 6-phosphate. This inhibition is not observed with mannose, glucose, fucose, glucose 6-phosphate, mannose 1-phosphate, galactose 1-phosphate, or galactose 6-phosphate. Preincubation of the effector cells, obtained from fresh whole blood, with mannose-6-phosphate, fructose-1-phosphate, or fructose-6-phosphate did not inhibit cytotoxicity, which indicated that these hexose phosphates are not nonspecifically toxic towards the effector lymphocytes. Mannose-6-phosphate and the stereochemically similar fructose-1-phosphate are more potent inhibitors than fructose-6-phosphate in terms of concentration required and time of onset of effect. Inhibition of cytotoxicity by mannose-6-phosphate varied with target cell type: F-265 is protected at much lower concentrations of mannose-6-phosphate (less than 1 mM) than is either Molt-4 or K-562. The inhibition of NCMC is also observed with the inhibitors of lysosomal function, NH4Cl, and chloroquine. The presence of a functional mannose-6-phosphate receptor on target cells was demonstrated: (i) Gelonin, a seed protein that inactivates the eukaryotic ribosome but is nontoxic to intact cells, was covalently linked to monophosphopentamannose, and this conjugate ws toxic to both K-562 and F-265 target cells, the latter being by far the more sensitive; and (ii) chloroquine, NH4Cl, and mannose-6-phosphate all inhibited the toxicity of gelonin-monophosphopentamannose. These results suggest either that a cytolytic lymphokine contains a hexose phosphate residue and may be taken up by target cells through the lysosomal/mannose 6-phosphate pathway or that such a residue is involved in target cell-effector cell recognition.
Cytotoxicity, Immunologic↗
Rates of enzyme-catalyzed exchange determined by two-dimensional NMR: a study of glucose 6-phosphate anomerization and isomerization.
The application of two-dimensional (2D) Fourier-transform NMR to the determination of rate constants of complex enzyme-catalyzed reactions in the steady state is described. The yeast phosphoglucose isomerase (EC 5.3.1.9)-catalyzed anomerization of glucose 6-phosphate (Glc-6-P) as well as its isomerization to fructose 6-phosphate (Fru-6-P) was chosen as an example. The 2D technique permitted the simultaneous monitoring of the time course of the anomerization and isomerization steps, from which the various reaction rates were determined. The results obtained in the steady state demonstrate the usefulness of the 2D technique by confirming that the anomerization of Glc-6-P is enzyme catalyzed and that the isomerization of the alpha anomer of Glc-6-P to Fru-6-P is at least 10 times faster than the isomerization of the beta anomer of Glc-6-P. These results are compared with reaction rates obtained by rapid-quench methods and the mechanistic implications are discussed. Extrapolation of these results suggests that the 2D Fourier-transform NMR method should be applicable in intact biological tissues.
Fourier Analysis↗
Inhibition by 2,5-anhydromannitol of glycolysis in isolated rat hepatocytes and in Ehrlich ascites cells.
2,5-Anhydromannitol decreases lactate formation and 3H2O formation from [5-3H]glucose in isolated rat hepatocytes metabolizing high concentrations of glucose. The inhibition of glycolysis is accompanied by a slight decrease in the cellular content of fructose-6-P and a more substantial decrease in the cellular content of fructose-1,6-P2, with no change in the content of glucose-6-P. The 3H2O release data and changes in hexosephosphate distribution indicate possible inhibitions at phosphofructokinase-1 and phosphoglucose isomerase. 2,5-Anhydromannitol also inhibits glycolysis in Ehrlich ascites cells, but the tumor cells, unlike hepatocytes, must be treated with 2,5-anhydromannitol prior to exposure to glucose to obtain the inhibition. The decrease in 3H2O formation from [5-3H]glucose and the metabolite pattern that results from the addition of low concentrations (less than or equal to 0.25 mM) of 2,5-anhydromannitol indicate an inhibition at phosphofructokinase-1 that cannot be attributed to a decrease in the cellular content of fructose-2,6-P2. Higher concentrations (greater than or equal to 0.5 mM) of 2,5-anhydromannitol cause a substantial decrease in the cellular content of ATP that is accompanied by decreases in the content of glucose-6-P and fructose-6-P and transient increases in fructose-1,6-P2. In Ehrlich ascites cells, 2,5-anhydromannitol is metabolized to 2,5-anhydromannitol mono- and bisphosphate. The inhibition of glycolysis caused by 2,5-anhydromanitol decreases with time, because the phosphorylated metabolites formed during the preliminary incubation in the absence of glucose are rapidly dephosphorylated during the incubation in the presence of glucose.
Animals↗
Crystal structure of the neutral form of fructose-1,6-bisphosphatase complexed with the product fructose 6-phosphate at 2.1-A resolution.
The crystal structure of fructose-1,6-bisphosphatase (EC 3.1.3.11) complexed with the product fructose 6-phosphate (F6P) has been refined at 2.1-A resolution to an R factor of 0.177 with root-mean-square deviations of 0.014 A and 2.9 degrees from the ideal geometries of bond lengths and bond angles, respectively. The secondary structures but not the trace of the unligated enzyme have been slightly revised in the F6P complex at this higher resolution. Helix H4 in the unligated structure has been refined to a helix-like coil, and two very short 3(10) helices have been found, one in H4 and one in H5. F6P at 10 mM concentration in the absence of divalent metals in our study shows major binding at the active site and minor binding at the AMP site. The major site has almost equal full occupancy in the C1 and C2 chains of the crystallographic asymmetric unit, while the minor site shows occupancy only in the C1 chain at about 50%. The electron density in both (2Fo - Fc) and (Fo - Fc) maps calculated by omitting F6P slightly favors the beta anomer of D-F6P over the alpha anomer. Possible functions of the active-site residues are discussed, and candidates are suggested for site-directed mutagenesis.
Adenosine Monophosphate↗
Allosteric transition of fructose-1,6-bisphosphatase.
Structural changes during the R-to-T transition of fructose-1,6-bisphosphatase (EC 3.1.3.11) form a hierarchy, in which structural changes at one level are supported by those at the other levels. The quaternary conformational changes involve a 17 degrees rotation between the upper and lower dimers, and a 3.4 degrees rotation between monomers in a dimer. Within monomers, the FBP domain, which remains rigid during the R-to-T transition, rotates 2.3 degrees relative to the AMP domain, which undergoes significant structural reorientations. The most important of these reorientations are the newly identified partially ordered loop residues 55-61 in the T state and reorientations of helices H1, H2, and H3. Supporting these structural changes are numerous readjustments of hydrogen bonding and van der Waals interactions throughout the entire tetrameric protein. Propagation of structural changes during the R-to-T transition relies primarily on helices H1, H2, H3, and loop 50-72. The change that begins at the AMP site causes reorientation of H1, H2, and H3 and changes of interactions across the C1-C4 (C2-C3) interface. These changes may propagate down H1, H2, H3, and loop 50-72 to affect interactions across the C1-C2 (C3-C4) and C1-C3 (C2-C4) interfaces. AMP inhibition is most probably caused by reduced metal binding affinity due to structural changes of metal ligands (Glu97, Asp118, and Asp121) in the active site. The eight-stranded beta-sheet, particularly the beta-strand B3, which connects Lys112 and Tyr113 of the AMP site with Asp118 and Asp121 of the metal site, may be responsible for communication between the AMP and active sites. Additional structural changes that support such communication include reorientation of the FBP domain and H1, H2, and H3 relative to the eight-stranded beta-sheet, and new conformations of loop 54-72 in the T state as AMP binds.
Adenosine Monophosphate↗