PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Fructosediphosphates”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources
PubMed1998

Conserved active site aspartates and domain-domain interactions in regulatory properties of the sugar kinase superfamily.

The structures of the sugar kinase/heat shock 70/actin superfamily of enzymes show that the active site is located in a deep cleft between two domains whose relative movement defines a domain closure conformational change thought to be involved in the catalytic and regulatory properties of members of the superfamily. To investigate the role of the domain closure in the regulatory behavior, site-directed mutagenesis is used to alter specific domain-domain interactions in Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase), a member of this superfamily. Two active site aspartate residues are conserved throughout the superfamily, one (Asp245 in glycerol kinase) which is proposed to act as a general base during catalysis and one (Asp10 in glycerol kinase) which interacts with the Mg(II) ion of the bound Mg(II)-nucleotide complex. Each of these residues participates in domain-domain interactions that are mediated by the bound substrates. The enzymes containing the substitutions Asp245 to Asn (D245N) or Asp10 to Asn (D10N) were purified by affinity chromatography, and the effects of the substitutions on the catalytic properties and regulation by the allosteric effectors, fructose 1,6-bisphosphate (FBP), and the glucose-specific phosphocarrier protein, IIIGlc (also known as IIAGlc), were determined. Each of the residues participates in catalysis; kcat/Katp is decreased 300-fold by the D245N substitution and 100-fold by the D10N substitution. Affinity labeling with the glycerol analog 1,3-dichloroacetone shows that the level of activity seen for the D245N mutant enzyme is not due to deamidation of the substituted asparagine. Each of the substitutions has little effect on regulation by FBP and the apparent affinity for IIIGlc, and the D245N substitution does not affect the extent of inhibition by IIIGlc. However, the D10N substitution decreases the maximum extent of inhibition by IIIGlc from 100 to 60%, thus changing the action of IIIGlc to that of a partial inhibitor. The different sensitivities of the extents of FBP and IIIGlc inhibition to perturbation of a domain-domain interaction mediated by Asp10 suggest that the relations of the actions of these allosteric effectors to the domain closure conformational change are different.

Allosteric Regulation↗
PubMed1998

A kinetic study of site-directed mutants of Escherichia coli ADP-glucose pyrophosphorylase: the role of residue 295 in allosteric regulation.

The effects of amino acid substitutions at residue 295 on the regulatory properties of Escherichia coli ADP-glucose pyrophosphorylase were studied. In previous studies, this residue, altered from proline to serine (P295S) in the gene of a mutant strain of E. coli, resulted in a high-activity form of enzyme [higher activity in absence of activator fructose 1,6-bisphosphate (FBP), higher apparent affinity for FBP and substrates, and lower apparent affinity for the inhibitor, AMP]. The effects of size and charge on this site were explored by replacing Pro with Gly, Asp, Asn, Gln, or Glu. All mutant enzymes were expressed and purified for kinetic analysis. All mutant enzymes, to varying extents, were in more active form than the wild-type enzyme. Enzymes with a substituted negative charge (P295D, P295E) had the highest activity in the absence of FBP, while the P295G enzyme was most similar to the wild type. The P295D and P295E enzymes had the lowest apparent affinities for AMP; this effect was partially abolished by the neutral substitutions P295N and P295Q. Another mutation, G336D, had previously been found to produce an even higher activity enzyme form. In order to examine interactions between substitutions at the 295 and 336 positions, the double mutant P295D-G336D was constructed and characterized. The double mutant enzyme was more active in the absence of FBP, with a higher affinity for FBP and a lower apparent affinity for AMP than either single mutated enzyme. The significance of residue 295 in regulation is discussed.

Adenosine Monophosphate↗
PubMed1999

cDNA sequence and kinetic properties of human lung fructose(1, 6)bisphosphatase.

A cDNA encoding fructose(1,6)bisphosphatase was isolated from total human lung RNA. The cDNA contained an open reading frame encoding 337 amino acids. The determined nucleotide sequence of the lung cDNA was significantly different from muscle cDNA and slightly differed from human liver cDNA in a single mutation (Gly-336 for Ala-336) and a T for C substitution in position 648. The human lung fructose(1, 6)bisphosphatase [Fru(1,6)Pase] was isolated and its kinetic parameters were compared with liver and muscle isoenzymes. Values of kcat for the lung Fru(1,6)Pase were lower than for the liver and muscle enzyme. Like the liver isoenzyme, lung Fru(1,6)Pase is significantly less inhibited by AMP than the muscle enzyme. The values of I0.5 were 9.5, 9.8, and 0.3 microM for the liver, lung, and muscle enzyme, respectively. The lung enzyme was slightly more sensitive to fructose(2,6)bisphosphate [Fru(2,6)P2] inhibition than the liver enzyme. Ki was 75 microM for the lung and 96 microM for the liver enzyme. The synergistic effect of AMP and Fru(2,6)P2 on the lung and liver Fru(1,6)Pase was also observed. In the presence of AMP the corresponding values of Ki for Fru(2,6)P2 were 16 microM for the lung and 10 microM for the liver enzyme.

Adenosine Monophosphate↗
PubMed1999

Kinetic characterization of a T-state of Ascaris suum phosphofructokinase with heterotropic negative cooperativity by ATP eliminated.

The affinity analogue, 2',3'-dialdehyde ATP has been used to chemically modify the ATP-inhibitory site of Ascaris suum phosphofructokinase, thereby locking the enzyme into a less active T-state. This enzyme form has a maximum velocity that is 10% that of the native enzyme in the direction of fructose 6-phosphate (F6P) phosphorylation. The enzyme displays sigmoid saturation for the substrate fructose 6-phosphate (S0.5 (F6P) = 19 mM and nH = 2.2) at pH 6.8 and a hyperbolic saturation curve for MgATP with a Km identical to that for the native enzyme. The allosteric effectors, fructose 2,6-bisphosphate and AMP, do not affect the S0.5 for F6P but produce a slight (1.5- and 2-fold, respectively) V-type activation with Ka values (effector concentration required for half-maximal activation) of 0.40 and 0.24 mM, respectively. Their activating effects are additive and not synergistic. The kinetic mechanism for the modified enzyme is steady-state-ordered with MgATP as the first substrate and MgADP as the last product to be released from the enzyme surface. The decrease in V and V/K values for the reactants likely results from a decrease in the equilibrium constant for the isomerization of the E:MgATP binary complex, thus favoring an unisomerized form. The V and V/KF6P are pH dependent with similar pK values of about 7 on the acid side and 9.8 on the basic side. The microenvironment of the active site appears to be affected minimally as evidenced by the similarity of the pK values for the groups involved in the binding site for F6P in the modified and native enzymes.

Adenosine Monophosphate↗
PubMed1999

Differential effect of glycolytic intermediaries upon cyclic ADP-ribose-, inositol 1',4',5'-trisphosphate-, and nicotinate adenine dinucleotide phosphate-induced Ca(2+) release systems.

We investigated the effect of glycolytic pathway intermediaries upon Ca(2+) release induced by cyclic ADP-ribose (cADPR), inositol 1',4', 5-trisphosphate (IP(3)), and nicotinate adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenate. Fructose 1,6, -diphosphate (FDP), at concentrations up to 8 mM, did not induce Ca(2+) release by itself in sea urchin egg homogenate. However, FDP potentiates Ca(2+) release mediated by agonists of the ryanodine channel, such as ryanodine, caffeine, and palmitoyl-CoA. Furthermore, glucose 6-phosphate had similar effects. FDP also potentiates activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca(2+) release was decreased approximately 3.5 times by addition of 4 mM FDP. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to FDP. The Ca(2+) release mediated by FDP in the presence of subthreshold concentrations of cADPR was inhibited by antagonists of the ryanodine channel, such as ruthenium red, and by the cADPR inhibitor 8-Br-cADPR. However, inhibition of Ca(2+) release induced by IP(3) or NAADP had no effect upon Ca(2+) release induced by FDP in the presence of low concentrations of cADPR. Furthermore, FDP had inhibitory effects upon Ca(2+) release induced by both IP(3) and NAADP. We propose that the state of cellular intermediary metabolism may regulate cellular Ca(2+) homeostases by switching preferential effects from one intracellular Ca(2+) release channel to another.

Adenosine Diphosphate Ribose↗
PubMed2000

Modulation of gluconeogenesis by epinephrine in hepatocytes isolated from genetically obese (fa/fa) Zucker rats.

The obese (fa/fa) Zucker rat shows an impaired sympathetic tone which is accompanied by an altered thermogenesis and changes in both lipid and carbohydrate metabolism. In this work, we have investigated the regulatory effects of epinephrine on the rate of gluconeogenesis from a mixture of [(14)C]lactate/pyruvate, in hepatocytes isolated from obese (fa/fa) rats and their lean (Fa/-) littermates. Epinephrine caused a dose-dependent stimulation of the rate of [(14)C]glucose formation in both obese and lean rat hepatocytes, the maximal rates being five- and twofold higher than the corresponding basal values (0.50 +/- 0.06 and 1.96 +/- 0.15 micromol of lactate converted to glucose/g of cell x 20 min, respectively). No significant differences were found between the calculated half-maximal effective concentrations (EC(50)) for epinephrine in obese and lean rat liver cells. The stimulation of gluconeogenesis by epinephrine was accompanied by a decrease in the cellular concentration of fructose 2,6-bisphosphate, and an inactivation of both pyruvate kinase and 6-phosphofructo 2-kinase, to similar extents in both types of hepatocytes. Epinephrine also significantly raised the hepatocyte content of cyclic AMP, with about a twofold increase at a saturating concentration of the catecholamine (1 microM), in both lean and obese rat liver cells. However, at suboptimal concentrations of epinephrine, the rise in cyclic AMP levels was significantly less marked in obese than in lean rat hepatocytes. Nevertheless, no significant differences were found in either the affinity or the number of beta-adrenergic receptors, in radioligand binding studies carried out in liver plasma membranes obtained from obese and lean Zucker rats. In conclusion, compared to the corresponding basal values, the response of gluconeogenesis from lactate to the stimulatory effect of epinephrine is higher in obese (fa/fa) than in lean (Fa/-) Zucker rat hepatocytes, with no significant differences in the calculated EC(50) values for this hormone. This occurs in spite of an apparent decreased sensitivity of the adenylate cyclase system to the stimulatory effect of epinephrine in obese rat liver cells.

Adenylyl Cyclases↗
PubMed2000

Lithium's effects on rat liver glucose metabolism in vivo.

Oral administration of lithium carbonate to fed-healthy rats strongly decreased liver glycogen content, despite the simultaneous activation of glycogen synthase and the inactivation of glycogen phosphorylase. The effect seemed to be related to a decrease in glucose 6-phosphate concentration and to a decrease in glucokinase activity. Moreover, in these animals lithium markedly decreased liver fructose 2,6-bisphosphate, which could be a consequence of the fall in glucose 6-phosphate and of the inactivation of 6-phosphofructo-2-kinase. Liver pyruvate kinase activity and blood insulin also decreased after lithium administration. Lower doses of lithium carbonate had less intense effects. Lithium administration to starved-healthy and fed-streptozotocin-diabetic rats caused a slight increase in blood insulin, which was simultaneous with increases in liver glycogen, glucose 6-phosphate, and fructose 2, 6-phosphate. Glucokinase, 6-phosphofructo-2-kinase, and pyruvate kinase activities also increased after lithium administration in starved-healthy and fed-diabetic rats. Lithium treatment activated glycogen synthase and inactivated glycogen phosphorylase in a manner similar to that observed in fed-healthy rats. Glycemia was not modified in any group of animals. These results indicate that lithium acts on liver glycogen metabolism in vivo in at least two different ways: one related to changes in insulinemia, and the other related to the direct action of lithium on the activity of some key enzymes of liver glucose metabolism.

Animals↗
PubMed2001

Photoaffinity labeling and photoaffinity cross-linking of phosphofructokinase-1 from Saccharomyces cerevisiae by 8-azidoadeninenucleotides.

Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four alpha- and four beta-subunits, each of them carrying catalytic and regulatory bindings sites for MgATP. In this paper, various photoaffinity labels, such as 8-azidoadenosine 5'-triphosphate, 8-azido-1,N6-ethenoadenosine 5'-triphosphate, and 8-N3-3'(2')-O-biotinyl-8-azidoadenosine 5'-triphosphate have been used to study their interaction with the enzyme in the dark and during irradiation. All nucleotidetriphosphates function as phosphate donor forming fructose 1,6-bisphosphate from fructose 6-phosphate. However, the kinetic analysis revealed distinctly differences between them. Photolabeling causes a decrease in enzyme activity to a similar extent, and ATP acts as competitive effector to inactivation. Three bifunctional diazidodiadeninedinucleotides (8-diN3AP4A, monoepsilon-8-diN3AP4A, and diepsilon-8-diN3AP4A) were applied for studying the spatial arrangement of the nucleotide binding sites. No cross-linking of the subunits was obtained by irradiation of the enzyme with 8-diN3AP4A. Photolabeling with diepsilon-8-diN3AP4A resulted in the formation of two alpha-beta cross-links with different mobilities in the SDS-polyacrylamide gel electrophoresis, while monoepsilon-8-diN3AP4A yielded only one alpha-beta cross-link. Because an interfacial location of the catalytic sites between two subunits is less likely, we suggest that the formation of cross-linked subunits may be the result of specific interactions of the bifunctional photolabels with regulatory sites at the interface of both subunits.

Adenosine Triphosphate↗
PubMed2002

Kinetic and allosteric consequences of mutations in the subunit and domain interfaces and the allosteric site of yeast pyruvate kinase.

The mechanism by which pyruvate kinase (PK) is allosterically activated by fructose-1,6-bisphosphate (FBP) is poorly understood. To identify residues key to allostery of yeast PK, a point mutation strategy was used. T403E and R459Q mutations in the FBP binding site caused reduced FBP affinity. Introducing positive charges at the 403, 458, and 406 positions in the FBP binding site had little consequence. The mutation Q299N in the A [bond] A subunit interface caused the enzyme response to ADP to be sensitive to FBP. The T311M A [bond] A interface mutant has a decreased affinity for PEP and FBP, and is dependent on FBP for activity. The R369A mutation in the C [bond] C interface only moderately influenced allostery. Creating an E392A mutation in the C [bond] C subunit interface eliminated all cooperativity and allosteric regulation. None of the seven A [bond] C domain interface mutations altered allostery. A model that includes a central role for E392 in allosteric regulation of yeast PK is proposed.

Adenosine Diphosphate↗
PubMed1993

A refinement of the Akabayashi-Saito-Kato modification of the enzymatic methods for 2-deoxyglucose and 2-deoxyglucose 6-phosphate.

Akabayashi et al. made a valuable modification of the enzymatic methods from our laboratory for measuring 2-deoxyglucose and 2-deoxyglucose 6-phosphate. Their modified procedure eliminates glucose and glucose 6-phosphate by conversion to fructose-1,6-bisphosphate, thereby saving two analytical steps. However, the present report describes a limitation of this new elimination procedure which is due to its unexpected reversibility, and provides an easy way to circumvent this limitation, namely heating to destroy the reagent enzymes before proceeding. The final result is a more flexible analytical scheme that is capable of measuring 2-deoxyglucose and its phosphate down to extremely low levels in the presence of up to thousandfold higher glucose concentrations. The completeness of glucose elimination eliminates both the problem of contamination of available glucose-6-phosphate dehydrogenases with 6-phosphogluconate dehydrogenase, and also the effect of the presence in this same enzyme of a trace of glucose dehydrogenase activity, which is an apparent side reaction.

Adenosine Diphosphate↗
PubMed1999

Automated sampling device for monitoring intracellular metabolite dynamics.

An automated sampling device coupled to a stirred tank reactor was developed for monitoring intracellular metabolite dynamics. Sample flasks fixed in transport magazines were moved by a step engine in a way that each sample flask was filled within 220 ms, resulting in a sampling rate of 4.5 s-1. Rapid inactivation of the metabolism was achieved by spraying the samples into 60% methanol at -50 degrees C. After centrifugation of the quenched cells at -20 degrees C the metabolites were extracted with perchloric acid and analyzed biochemically or with HPLC. The automated sampling device was applied for investigation of the intracellular metabolite dynamics of glycolysis in Escherichia coli after rapid glucose addition to a glucose-limited steady-state culture. For the first time oscillations of intracellular metabolite concentrations like glucose-6-phosphate, phosphoenolpyruvate, glyceraldehyde 3-phosphate, dihydroxyacetonphosphate, 3-phosphoglycerate, and pyruvate were quantified on a subseconds to seconds scale in E. coli. As an example, the kinetics of the decomposition of fructose 1, 6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetonphosphate were investigated by use of a well-known mechanistic kinetic model and the measured in vivo metabolite dynamics.

Biosensing Techniques↗
PubMed2001

Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques.

The quantitative comprehension of microbial metabolic networks is a prerequisite for an efficient rational strain improvement ("metabolic engineering"). It is therefore necessary to accurately determine the concentration of a large number of reactants (i.e., metabolites, nucleotides, cofactors) in order to understand "in vivo" reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates and nucleotides in Escherichia coli K12 using a perchloric acid extraction and an LC-ESI-MS method was achieved. Intracellular metabolites (e.g., glucose 6-phosphate, fructose 1,6-bisphosphate, 6-phospho gluconate, acetyl-CoA, adenine nucleotides) were quantified under defined (glucose-limited steady-state) growth conditions. The method was verified by comparing the intracellular metabolite concentrations measured via LC-ESI-MS with enzymatic determinations. It is thus possible to identify and quantify more than 15 intracellular metabolites in parallel with a minimal amount of sample volume.

Acetyl Coenzyme A↗
PubMed1995

Allosteric regulation of eukaryotic initiation factor eIF-2B by adenine nucleotides.

Previous studies have shown that eIF-2B purified from rabbit reticulocytes binds ATP and that the binding is prevented by NADP+. Because NADP+ inhibits the activity of eIF-2B in in vitro reactions we have examined whether or not the activity of eIF-2B is modulated by ATP. In these studies, eIF-2B, purified from rat liver, was incubated with ATP prior to assay. We found that the activity of eIF-2B was inhibited with an IC50 of approximately 0.8 mM. The inhibition was not due to phosphorylation of the factor. However, the inhibition of eIF-2B activity caused by ATP could be prevented by coincubation with either NADPH or fructose-1,6-bisphosphate. The activity of eIF-2B was also inhibited following addition of either ATP or AMPPNP to a post-mitochondrial supernatant prepared from rat liver. Therefore, it is possible that the activity of eIF-2B might be allosterically regulated in vivo not only by changes in the redox state of pyridine dinucleotides but also by changes in the relative amounts of NADPH and ATP.

Adenine Nucleotides↗
PubMed1995

Isozyme analysis of human normal polymorphonuclear leukocyte phosphofructokinase.

Phosphofructokinase (PFK) from human polymorphonuclear leukocytes (PMN) was characterized by immunological titration with subunit specific antibodies, column chromatography on QAE-Sephadex and SDS-polyacrylamide gel electrophoresis. Two different isozymes, M-type and L-type, were found. The M(r) values of the M and L subunits were 79,500 +/- 1,914 and 74,250 +/- 1,258, respectively. The two isozymes presented different kinetic and regulatory properties. The results suggest that PFK from human normal PMN is a mixture of M-type and L-type homotetramers, mainly, with possible minor heterotetrameric forms.

Adenosine Triphosphate↗
PubMed1995

Allosteric activation of rabbit reticulocyte guanine nucleotide exchange factor activity by sugar phosphates and inositol phosphates.

Sugar phosphates are required to maintain active rates of translation in gel-filtered rabbit reticulocyte lysates. They may stimulate polypeptide chain initiation by acting as NADPH generators or by a direct interaction with initiation factor(s). We now provide evidence for the allosteric activation of the purified guanine nucleotide exchange factor (eIF-2B) by sugar phosphates and inositol phosphates. In the presence of microM fructose 1,6-bisphosphate, the rate of eIF-2B-catalyzed GDP/GTP exchange is increased approximately 2-fold. The half-maximal concentration for stimulation of eIF-2B activity (SC50) is 57 microM. The binding of GTP to isolated eIF-2B is stimulated 1.5-fold, whereas GTP-binding to ALP-treated eIF-2B is not affected by sugar phosphates. Inositol 1,4-bisphosphate, like fructose 1,6-bisphosphate, stimulates 2-3-fold the activity of the isolated eIF-2B (SC50, 140 microM).

Allosteric Regulation↗
PubMed1996

A mechanism of regulation of hepatic Fru 2,6-P2 concentration upon refeeding: involvement of xylulose 5-P and cyclic-AMP.

In order to determine the mechanism for delayed increase in Fructose 2,6-P2 in livers of refed rats, the time course of changes in various metabolites upon refeeding NIH or high sucrose diet was investigated. Kinetics of increase in Fructose 2,6-P2 and Xylulose 5-P were similar but different from hexose 6-P or glycogen in the livers of 48 h starved rats refed with NIH diet. The increase in the Fructose 2,6-P2 level was a result of a combination of changes in Fructose 6-P,2-kinase and Fructose 2,6-bisphosphatase activity ratios, indicating dephosphorylation of the bifunctional enzyme and decreased cAMP. A similar correlation between Fructose 2,6-P2 and Xylulose 5-P and dephosphorylation was observed with refeeding high sucrose diet and also with 16 h starved rats. These kinetic results are consistent with the idea that a specific protein phosphatase 2A, activated by Xylulose 5-P, dephosphorylates Fructose 6-P,2-kinase:Fructose 2,6-bisphosphatase and also decreased protein kinase A activity, resulting in increased hepatic Fructose 2,6-P2.

Animals↗
PubMed1996

Isozyme analysis of human polymorphonuclear leukocyte phosphofructokinase from insulin resistant individuals.

Phosphofructokinase (PFK) from human polymorphonuclear leukocytes (PMN) was characterized by immunological titration with subunit specific antibodies and column chromatography on QAE-Sephadex in three different groups: control, type II diabetic, and obese individuals. It was found that PMN phosphofructokinase in the three groups consists mainly of a mixture of L4 and M4 homotetramers with possibly some hybrid forms. The predominant subunit was the L-type. A 24% decrease in the specific activity of the L-type isozyme was observed and an intermediate form (I-isozyme) having 23% of the total activity in diabetic individuals appeared. In obese individuals a 30% decrease was observed in the activity of M-type isozyme and 9% of the total activity corresponded to the intermediate form. Kinetic studies showed different regulatory properties among the isozymes from the three groups. The lower PFK activity found in diabetic and obese individuals can be associated with the decreased activity in the L-type isozyme (for diabetic individuals) and in the M-type isozyme (for obese individuals); the lower activity can also be associated with the four times lower affinity for F-6-P showed by the M-type isozyme, the decreased sensitivity to ATP inhibition (for both isozymes), and the appearance of an intermediate form with a different kinetic behaviour.

Adenosine Triphosphate↗