The rate of substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate in skeletal muscle from cold-exposed, hyperthyroid or acutely exercised rats.
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ABSTRACT:
SUBMITTER: Challis RA
PROVIDER: S-EPMC1152728 | biostudies-other | 1985 Oct
REPOSITORIES: biostudies-other
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Project description:Substrate cycling of fructose 6-phosphate through reactions catalysed by 6-phosphofructokinase and fructose-1,6-bisphosphatase was measured in skeletal muscles of the rat in vitro. The rate of this cycle was calculated from the steady-state values of the 3H/14C ratio in hexose monophosphates and fructose 1,6-bisphosphate after the metabolism of either [5-3H,6-14C]glucose or [3-3H,2-14C] glucose. Two techniques for the separation of hexose phosphates were studied; t.l.c. chromatography on poly(ethyleneimine)-cellulose sheets or ion-exchange chromatography coupled with enzymic conversion. These two methods gave almost identical results, suggesting that either technique could be used for determination of rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling. It was found that more than 50% of the 3H was retained in the fructose 1,6-bisphosphate; it is therefore probable that previous measurement of cycling rates, which have assumed complete loss of 3H, have underestimated the rate of this cycle. The effects of insulin, adrenaline and adrenergic agonists and antagonists on rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling were investigated. In the presence of insulin, adrenaline (1 microM) increased the cycling rate by about 10-fold in epitrochlearis muscle in vitro; the maximum rate under these conditions was about 2.5 mumol/h per g of tissue. The concentration of adrenaline that increased the cycling rate by 50% was about 50 nM. This effect of adrenaline appears to be mediated by the beta-adrenergic receptor, since the rate was increased by beta-adrenergic agonists and blocked by beta-adrenergic antagonists. From the knowledge of the precise rate of this cycle, the possible physiological importance of cycling is discussed.
| S-EPMC1144014 | biostudies-other
Project description:The effects of several agents on the rates of the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle were measured in incubated epitrochlearis muscles of the rat by monitoring the transfer of radiolabel from [6-14C]glucose to the 1-position of glucose residues in glycogen. The cycling rates observed were almost identical with those previously obtained by using the well-established dual-isotope technique. In particular, it was found that the beta-adrenoceptor agonist isoprenaline increased the cycling rate about 12-fold.
| S-EPMC1144371 | biostudies-other
Project description:Fructose bisphosphate aldolase (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources, including the bacterium Brucella melitensis and the protozoan Babesia bovis. Bioinformatic analysis of the Bartonella henselae genome revealed an FBPA homolog. The B. henselae FBPA enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme crystallized in the apo form but failed to diffract; however, well diffracting crystals could be obtained by cocrystallization in the presence of the native substrate fructose 1,6-bisphosphate. A data set to 2.35 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=72.39, b=127.71, c=157.63 Å. The structure was refined to a final free R factor of 22.2%. The structure shares the typical barrel tertiary structure and tetrameric quaternary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site.
| S-EPMC3169401 | biostudies-literature
Project description:The apicomplexan parasite Toxoplasma gondii must invade host cells to continue its lifecycle. It invades different cell types using an actomyosin motor that is connected to extracellular adhesins via the bridging protein fructose-1,6-bisphosphate aldolase. During invasion, aldolase serves in the role of a structural bridging protein, as opposed to its normal enzymatic role in the glycolysis pathway. Crystal structures of the homologous Plasmodium falciparum fructose-1,6-bisphosphate aldolase have been described previously. Here, T. gondii fructose-1,6-bisphosphate aldolase has been crystallized in space group P22121, with the biologically relevant tetramer in the asymmetric unit, and the structure has been determined via molecular replacement to a resolution of 2.0?Å. An analysis of the quality of the model and of the differences between the four chains in the asymmetric unit and a comparison between the T. gondii and P. falciparum aldolase structures is presented.
| S-EPMC4157416 | biostudies-literature
Project description:Fructose 1,6-bisphosphate aldolase is a ubiquitous cytosolic enzyme that catalyzes the fourth step of glycolysis. Aldolases are classified into three groups: Class-I, Class-IA, and Class-II; all classes share similar structural features but low amino acid identity. Apart from their conserved role in carbohydrate metabolism, aldolases have been reported to perform numerous non-enzymatic functions. Here we review the myriad "moonlighting" functions of this classical enzyme, many of which are centered on its ability to bind to an array of partner proteins that impact cellular scaffolding, signaling, transcription, and motility. In addition to the cytosolic location, aldolase has been found the extracellular surface of several pathogenic bacteria, fungi, protozoans, and metazoans. In the extracellular space, the enzyme has been reported to perform virulence-enhancing moonlighting functions e.g., plasminogen binding, host cell adhesion, and immunomodulation. Aldolase's importance has made it both a drug target and vaccine candidate. In this review, we note the several inhibitors that have been synthesized with high specificity for the aldolases of pathogens and cancer cells and have been shown to inhibit classical enzyme activity and moonlighting functions. We also review the many trials in which recombinant aldolases have been used as vaccine targets against a wide variety of pathogenic organisms including bacteria, fungi, and metazoan parasites. Most of such trials generated significant protection from challenge infection, correlated with antigen-specific cellular and humoral immune responses. We argue that refinement of aldolase antigen preparations and expansion of immunization trials should be encouraged to promote the advancement of promising, protective aldolase vaccines.
| S-EPMC8385298 | biostudies-literature
Project description:Several 5-O-alkyl- and 5-C-alkyl-mannitol bis-phosphates were synthesized and comparatively assayed as inhibitors of fructose bis-phosphate aldolases (Fbas) from rabbit muscle (taken as surrogate model of the human enzyme) and from Trypanosoma brucei. A limited selectivity was found in several instances. Crystallographic studies confirm that the 5-O-methyl derivative binds competitively with substrate and the 5-O-methyl moiety penetrating deeper into a shallow hydrophobic pocket at the active site. This observation can lead to the preparation of selective competitive or irreversible inhibitors of the parasite Fba.
| S-EPMC4018070 | biostudies-literature
Project description:Yeast and cancer cells share the unusual characteristic of favoring fermentation of sugar over respiration. We now reveal an evolutionary conserved mechanism linking fermentation to activation of Ras, a major regulator of cell proliferation in yeast and mammalian cells, and prime proto-oncogene product. A yeast mutant (tps1?) with overactive influx of glucose into glycolysis and hyperaccumulation of Fru1,6bisP, shows hyperactivation of Ras, which causes its glucose growth defect by triggering apoptosis. Fru1,6bisP is a potent activator of Ras in permeabilized yeast cells, likely acting through Cdc25. As in yeast, glucose triggers activation of Ras and its downstream targets MEK and ERK in mammalian cells. Biolayer interferometry measurements show that physiological concentrations of Fru1,6bisP stimulate dissociation of the pure Sos1/H-Ras complex. Thermal shift assay confirms direct binding to Sos1, the mammalian ortholog of Cdc25. Our results suggest that the Warburg effect creates a vicious cycle through Fru1,6bisP activation of Ras, by which enhanced fermentation stimulates oncogenic potency.Yeast and cancer cells both favor sugar fermentation in aerobic conditions. Here the authors describe a conserved mechanism from yeast to mammals where the glycolysis intermediate fructose-1,6-bisphosphate binds Cdc25/Sos1 and couples increased glycolytic flux to increased Ras proto-oncoprotein activity.
| S-EPMC5640605 | biostudies-literature
Project description:Glycolysis is one of the important ways by which Echinococcus multilocularis acquires energy. Fructose-1, 6-bisphosphate aldolase (FBA) plays an important role in this process, but it is not fully characterized in E. multilocularis yet. The results of genome-wide analysis showed that the Echinococcus species contained four fba genes (FBA1-4), all of which had the domain of FBA I and multiple conserved active sites. EmFBA1 was mainly located in the germinal layer and the posterior of the protoscolex. The enzyme activity of EmFBA1 was 67.42 U/mg with Km and Vmax of 1.75 mM and 0.5 mmol/min, respectively. EmFBA1 was only susceptible to Fe3+ but not to the other four ions (Na+, Ca2+, K+, Mg2+), and its enzyme activity was remarkably lost in the presence of 0.5 mM Fe3+. The current study reveals the biochemical characters of EmFBA1 and is informative for further investigation of its role in the glycolysis in E. multilocularis.
| S-EPMC8781991 | biostudies-literature
Project description:The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK), but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex containing at least v-ATPase, ragulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts the association of axin and LKB1 with v-ATPase and ragulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.
| S-EPMC5544942 | biostudies-literature
Project description:Fructose-1,6-bisphosphate (FBP) aldolase, a glycolytic enzyme, catalyzes the reversible and stereospecific aldol addition of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (d-G3P) by an unresolved mechanism. To afford insight into the molecular determinants of FBP aldolase stereospecificity during aldol addition, a key ternary complex formed by DHAP and d-G3P, comprising 2% of the equilibrium population at physiological pH, was cryotrapped in the active site of Toxoplasma gondii aldolase crystals to high resolution. The growth of T. gondii aldolase crystals in acidic conditions enabled trapping of the ternary complex as a dominant population. The obligate 3(S)-4(R) stereochemistry at the nascent C3-C4 bond of FBP requires a si-face attack by the covalent DHAP nucleophile on the d-G3P aldehyde si-face in the active site. The cis-isomer of the d-G3P aldehyde, representing the dominant population trapped in the ternary complex, would lead to re-face attack on the aldehyde and yield tagatose 1,6-bisphosphate, a competitive inhibitor of the enzyme. We propose that unhindered rotational isomerization by the d-G3P aldehyde moiety in the ternary complex generates the active trans-isomer competent for carbonyl bond activation by active-site residues, thereby enabling si-face attack by the DHAP enamine. C-C bond formation by the cis-isomer is suppressed by hydrogen bonding of the cis-aldehyde carbonyl with the DHAP enamine phosphate dianion through a tetrahedrally coordinated water molecule. The active site geometry further suppresses C-C bond formation with the l-G3P enantiomer of d-G3P. Understanding C-C formation is of fundamental importance in biological reactions and has considerable relevance to biosynthetic reactions in organic chemistry.
| S-EPMC5712624 | biostudies-literature