Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.2.13 (aldolase)
3,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13) have been highly purified from Saccharomyces cerevisiae by improved protocols. Partitioning of the enzymes in aqueous polymer two-phase systems was used to detect complex formation. The partition of each enzyme was found to be affected by the presence of the other enzyme. AMP affected the partition of the individual enzymes as well as the mixture of the two. The activities of the respective enzymes were stimulated in the putative complex in an AMP-dependent manner. Two strictly conserved residues belonging to an acidic surface loop of class II aldolases, are a potential site for electrostatic interaction with the positively charged regions close to the active site in phosphofructokinase.
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PMID:Interaction between phosphofructokinase and aldolase from Saccharomyces cerevisiae studied by aqueous two-phase partitioning. 1123 90

The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIA(Glc), as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIA(Glc) phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIA(Glc). Isopropyl-beta-D-thiogalactopyranoside-induced overexpression of EIIA(Glc) did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIA(Glc). A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIA(Glc) is controlled by G3P and other phosphorylated sugars such as D-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and D-tagatose-1,6-bisphosphate aldolase.
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PMID:Glycerol-3-phosphate-induced catabolite repression in Escherichia coli. 1200 46

Phosphorylated fructose-1,6-bisphosphatase (FBPase) was isolated from rabbit muscle in an SDS/PAGE homogeneous form. Its dephosphorylation with alkaline phosphatase revealed 2.8 moles of inorganic phosphate per mole of FBPase. The phosphorylated FBPase (P-FBPase) differs from the dephosphorylated enzyme in terms of its kinetic properties like K(m) and k(cat), which are two times higher for the phosphorylated FBPase, and in the affinity for aldolase, which is three times lower for the dephosphorylated enzyme. Dephosphorylated FBPase can be a substrate for protein kinase A and the amount of phosphate incorporated per FBPase monomer can reach 2-3 molecules. Since interaction of muscle aldolase with muscle FBPase results in desensitisation of the latter toward AMP inhibition (Rakus & Dzugaj, 2000, Biochem. Biophys. Res. Commun. 275, 611-616), phosphorylation may be considered as a way of muscle FBPase activity regulation.
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PMID:Rabbit muscle fructose-1,6-bisphosphatase is phosphorylatedin vivo. 1267 51

Real-time interaction analysis, using the BIAcore biosensor, of rabbit muscle FBPase-aldolase complex revealed apparent binding constant [K(Aapp)] values of about 4.4x10(8) M(-1). The stability of the complex was down-regulated by the glycolytic intermediates dihydroxyacetone phosphate and fructose 6-phosphate, and by the regulator of glycolysis and glyconeogenesis--fructose 2,6-bisphosphate. FBPase in a complex with aldolase was entirely insensitive to inhibition by physiological concentrations of AMP (I(0.5) was 1.35 mM) and the cooperativity of the inhibition was not observed. The existence of an FBPase-aldolase complex that is insensitive to AMP inhibition explains the possibility of glycogen synthesis from carbohydrate precursors in vertebrates' myocytes.
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PMID:Muscle FBPase in a complex with muscle aldolase is insensitive to AMP inhibition. 1286 Mar 78

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) is strongly inhibited by AMP in vitro and, therefore, at physiological concentrations of substrate and AMP, FBPase should be completely inhibited. Desensitization of rabbit muscle FBPase against AMP inhibition was previously observed in the presence of rabbit muscle aldolase. In this study, we analysed the kinetics of an FBPase catalyzed reaction and interaction between chicken muscle FBPase and chicken muscle aldolase. The initial rate of FBPase reaction vs. substrate concentration shows a maximum activity at a concentration of 20 microM Fru-1,6P2 and then decreases. Assuming rapid equilibrium kinetics, the enzyme-catalyzed reaction was described by the substrate inhibition model, with Ks approximately 5 microM and Ksi approximately 39 microM and factor beta approximately 0.2, describing change in the rate constant (k) of product formation from the ES and ESSi complexes. Based on ultracentrifugation studies, aldolase and FBPase form a hetero-complex with approximately 1:1 stoichiometry with a dissociation constant (Kd) of 3.8 microM. The FBPase-aldolase interaction was confirmed via fluorescence investigation. The aldolase-FBPase interaction results in aldolase fluorescence quenching and its maximum emission spectrum shifting from 344 to 356 nm. The Kd of the FBPase-aldolase complex, determined on the basis of fluorescence changes, is 0.4 microM at 25 degrees C with almost 1:1 stoichiometry. This interaction increases the I(0.5) for the AMP inhibition of FBPase threefold, and slightly affects FBPase affinity to magnesium ions, increasing the Ka and Hill coefficient (n). No effect of aldolase on the FBPase pH optimum was observed. Thus, the decrease in FBPase sensitivity to AMP inhibition enables FBPase to function in vivo thanks to aldolase.
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PMID:The interaction of FBPase with aldolase: a kinetic and fluorescence investigation on chicken muscle enzymes. 1469 18

The nature of oxidative damage to Saccharomyces cerevisiae caused by levels of HOCl that inhibit cell replication was explored with the intent of identifying the loci of lethal lesions. Functions of cytosolic enzymes and organelles that are highly sensitive to inactivation by HOCl, including aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the mitochondrion, were only marginally affected by exposure of the yeast to levels of HOCl that completely inhibited colony formation. Loss of function in membrane-localized proteins, including the hexose transporters and PMA1 H(+)-ATPase, which is the primary proton pump located within the S. cerevisiae plasma membrane, was also marginal and K(+) leak rates to the extracellular medium increased only slowly with exposure to increasing amounts of HOCl, indicating that the plasma membrane retained its intrinsic impermeability to ions and metabolites. Adenylate phosphorylation levels in fermenting yeast declined in parallel with viability; however, yeast grown on respiratory substrates maintained near-normal phosphorylation levels at HOCl doses several-fold greater than that required for killing. This overall pattern of cellular response to HOCl differs markedly from that previously reported for bacteria, which appear to be killed by inhibition of plasma membrane proteins involved in energy transduction. The absence of significant loss of function in critical oxidant-sensitive cellular components and retention of ATP-synthesizing capabilities in respiring yeast cells exposed to lethal levels of HOCl suggests that toxicity in this case may arise by programmed cell death.
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PMID:HOCl-mediated cell death and metabolic dysfunction in the yeast Saccharomyces cerevisiae. 1487 79

Fructose 1,6-bisphosphatase (FBPase) is known to form a supramolecular complex with alpha-actinin and aldolase on both sides of the Z-line in skeletal muscle cells. It has been proposed that association of aldolase with FBPase not only desensitizes muscle FBPase toward AMP inhibition but it also might enable the channeling of intermediates between the enzymes [Rakus et al. (2003) FEBS Lett. 547, 11-14]. In the present paper, we tested the possibility of fructose 1,6-bisphosphate (F1,6-P(2)) channeling between aldolase and FBPase using the approach in which an inactive form of FBPase competed with active FBPase for binding to aldolase and thus decreased the rate of aldolase-FBPase reaction. The results showed that F1,6-P(2) is transferred directly from aldolase to FBPase without mixing with the bulk phase. Further evidence that F1,6-P(2) is channeled from aldolase to FBPase comes from the experiments investigating the inhibitory effect of a high concentration of magnesium ions on aldolase-FBPase activity. FBPase in a complex with aldolase, contrary to free muscle FBPase, was not inhibited by high Mg(2+) concentrations, which suggests that free F1,6-P(2) was not present in the assay mixture during the reaction. A real-time interaction analysis between aldolase and FBPase revealed a dual role of Mg(2+) in the regulation of the aldolase-FBPase complex stability. A physiological concentration of Mg(2+) increased the affinity of muscle FBPase to muscle aldolase, whereas higher concentrations of the cation decreased the concentration of the complex. We hypothesized that the presence of Mg(2+) stabilizes a positively charged cavity within FBPase and that it might enable an interaction with aldolase. Because magnesium decreased the binding constant (K(a)) between aldolase and FBPase in a manner similar to the decrease of K(a) caused by monovalent cations, it is postulated that electrostatic attraction might be a driving force for the complex formation. It is presumed that the biological relevance of F1,6-P(2) channeling between aldolase and FBPase is protection of this glyconeogenic, as well as glycolytic, intermediate against degradation by cytosolic aldolase, which is one of the most abundant enzyme of glycolysis.
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PMID:Interaction between muscle aldolase and muscle fructose 1,6-bisphosphatase results in the substrate channeling. 1555 2

Cytosolic fructose-1,6-P(2) (FBP) aldolase (ALD(c)) from germinated mung beans has been purified 1078-fold to electrophoretic homogeneity and a final specific activity of 15.1 micromol FBP cleaved/min per mg of protein. SDS-PAGE of the final preparation revealed a single protein-staining band of 40 kDa that cross-reacted strongly with rabbit anti-(carrot ALD(c))-IgG. The enzyme's native M(r) was determined by gel filtration chromatography to be 160 kDa, indicating a homotetrameric quaternary structure. This ALD is a class I ALD, since EDTA or Mg(2+) had no effect on its activity, and was relatively heat-stable losing 0-25% of its activity when incubated for 5 min at 55-65 degrees C. It demonstrated: (i) a temperature coefficient (Q(10)) of 1.7; (ii) an activation energy of 9.2 kcal/mol active site; and (iii) a broad pH-activity optima of 7.5. Mung bean ALD(c) is bifunctional for FBP and sedoheptulose-1,7-P(2) (K(m) approximately 17 microM for both substrates). ATP, ADP, AMP and ribose-5-P exerted inhibitory effects on the activity of the purified enzyme. Ribose-5-P, ADP and AMP functioned as competitive inhibitors (K(i) values=2.2, 3.1 and 7.5mM, respectively). By contrast, the addition of 2mM ATP: (i) reduced V(max) by about 2-fold, (ii) increased K(m)(FBP) by about 4-fold, and (iii) shifted the FBP saturation kinetic plot from hyperbolic to sigmoidal (h=1.0 and 2.6 in the absence and presence of 2mM ATP, respectively). Potent feedback inhibition of ALD(c) by ATP is suggested to help balance cellular ATP demands with the control of cytosolic glycolysis and respiration in germinating mung beans.
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PMID:Purification and characterization of an allosteric fructose-1,6-bisphosphate aldolase from germinating mung beans (Vigna radiata). 1589 64

N-terminal residues of muscle fructose 1,6-bisphosphatase (FBPase) are highly conserved among vertebrates. In this article, we present evidence that the conservation is responsible for the unique properties of the muscle FBPase isozyme: high sensitivity to AMP and Ca(2+) inhibition and the high affinity to muscle aldolase, which is a factor desensitizing muscle FBPase toward AMP and Ca(2+). The first N-terminal residue affecting the affinity of muscle FBPase to aldolase is arginine 3. On the other hand, the first residue significantly influencing the kinetics of muscle FBPase is proline 5. Truncation from 5-7 N-terminal residues of the enzyme not only decreases its affinity to aldolase but also reduces its k-(cat) and activation by Mg(2+), and desensitizes FBPase to inhibition by AMP and calcium ions. Deletion of the first 10 amino acids of muscle FBPase abolishes cooperativity of Mg(2+) activation and results in biphasic inhibition of the enzyme by AMP. Moreover, this truncation lowers affinity of muscle FBPase to aldolase about 14 times, making it resemble the liver isozyme. We suggest that the existence of highly AMP-sensitive muscle-like FBPase, activity of which is regulated by metabolite-dependent interaction with aldolase enables the precise regulation of muscle energy expenditures and might contributed to the evolutionary success of vertebrates.
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PMID:Evolutionary conserved N-terminal region of human muscle fructose 1,6-bisphosphatase regulates its activity and the interaction with aldolase. 1821 67

The present work describes the selective covalent modification of fructose bisphosphate aldolase in crude extracts of chicken breast muscle by fluorescein 5'-isothiocyanate (5'-FITC) at pH 7.0 and 35 degrees C. The modification was observed after 1 min while no other major soluble protein was labeled even after 30 min. We calculated that ca. one 5'-FITC molecule was incorporated into each aldolase tetramer after a 30 min reaction which resulted in a minimal loss of enzyme activity. The "native" structure of aldolase was required for the selective modification by 5'-FITC since high pH, high temperature, and ionic detergents either inhibited or prevented the reaction of 5'-FITC with aldolase. Certain metabolites (ATP, ADP, CTP, GTP, FBP) and erythrosin B also inhibited the 5'-FITC modification of aldolase. In contrast, F-6-P, AMP, NADH, and NAD(+) as well as free lysine and most importantly, the 6'-isomer of FITC exhibited no competition with 5'-FITC for the labeling of aldolase. Alone, the 6'-isomer of FITC did not exhibit preferential reaction when combined with aldolase. 5'-FITC-labeled and -unlabeled aldolases were not distinguished by their ability to bind to muscle myofibrils (MFs) or by their abilities to refold following reversible denaturation in urea. Structural analysis revealed that 5'-FITC-labeled a tryptic peptide corresponding to residues 112-134 in the primary structure of aldolase, a peptide that does not contain lysine, the amino acid believed to be the primary target of this reagent. Unlike chicken and rabbit muscle aldolases, chicken brain and liver aldolase isoforms along with several other aldolases derived from diverse biological sources did not exhibit this highly selective modification by 5'-FITC.
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PMID:A selective reaction of fructose bisphosphate aldolase with fluorescein isothiocyanate in chicken muscle extracts. 1843 70


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