Initiation of translation at a UAG stop codon in the aldolase gene of Plasmodium falciparum.

The gene coding for the key glycolytic enzyme fructose-1,6-diphosphate aldolase of the human malaria parasite Plasmodium falciparum lacks a functional AUG initiation codon for translation. Protein sequences of natural or in vitro translated aldolase include the candidate start methionine residue at internal positions. No additional AUG start codon is found in genomic DNA, cDNA or mRNA sequences. Instead, a UAG chain termination codon is recognized as the start signal of protein synthesis in vivo and in vitro.     

Rapid cessation of phospholipid synthesis in fructose-1,6-diphosphate aldolase mutants of Escherichia coli.

Escherichia coli GH352, which was originally described as a temperature-sensitive strain containing a thermolabile acyl coenzyme A:monoacylglycerol 3-phosphate acyltransferase, does not now contain a thermolabile form of this enzyme. It has a defect in fructose-1,6-diphosphate aldolase and at least one additional temperature-sensitive lesion. Both strains GH352 and NP315, a temperature-sensitive aldolase mutant, show rapid cessation of 32-P1 incorporation into nucleic acids and phospholipids at 42 C. These characteristics of strain GH352 are therefore no longer attributed to thermolabile phospholipid synthesis, but can be attributed to the fructose-1,6-diphophate aldolase lesion.     

Liver aldolase as a possible target for the action of N-methyl-N-nitrosourea

The effect of N-methyl-N-nitrosourea (MNU) on the activity of cytoplasmic and reversibly bound to subcellular structures liver aldolase was studied. In vitro, the activity of aldolase purified from rabbit muscles is inhibited by MNU by 70-80% relative to fructose-1,6-diphosphate and by 50-60% relative to fructose-1-phosphate. These substrates and the competitive inhibitor ATP do not protect the enzyme against the inactivation by MNU. MNU inhibits the activity of cytoplasmic aldolase by 30-40% and 20% 2-24 hours after a single injection (80 mg/kg) in vivo. The enzyme affinity for fructose-1,6-diphosphate is markedly decreased (2-fold). Activation of cytoplasmic aldolase relative to both substrates, which is especially well-pronounced with fructose-1-phosphate after inhibition of the enzyme activity, was observed. The enzyme activity relative to both substrates was found to increase in the mitochondrial and nuclear fractions within 48 hours. MNU has no effect on the activity of aldolase bound to microsomes. MNU influences the aldolase binding to organelle membranes. MNU injections at early periods (2-168 hours) accounts for the differences in the kinetic properties of cytoplasmic and reversibly bound to subcellular structures liver aldolase. These changes persist within 168 hours after MNU administration and may result in disturbances in cell metabolism as well as in the regulation of metabolic pathways, such as glycolysis and gluconeogenesis.     

Neurospora fructose-1,6-diphosphate aldolase: inhibition by sodium pyruvate

$\text{Neurospora}$ fructose-1,6-diphosphate aldolase exhibited a hyperbolic substrate saturation curve which changed to sigmoidal in the presence of 0.5 mM sodium pyruvate. The S_0.5 value for fructose-1,6-diphosphate increased from 1.4 mM to 6.6 and 20 mM in the presence of 0.5 and 1.0 mM sodium pyruvate, respectively. The inhibition seems to be cooperative in nature and involves conformational changes. Potassium ions completely blocked the inhibition by sodium pyruvate.     

Isolation of a Mutant of Escherichia coli with a Temperature-sensitive Fructose-1,6-Diphosphate Aldolase Activity

B?ck, August (Purdue University, Lafayette, Ind.), and Frederick C. Neidhardt. Isolation of a mutant of Escherichia coli with a temperature-sensitive fructose-1,6-diphosphate aldolase activity. J. Bacteriol. 92:464-469. 1966.-A mutant of Escherichia coli was isolated which was able to grow in rich medium at 30 C but not at 40 C. Upon exposure to 40 C, the cells immediately stopped ribonucleic acid (RNA) and deoxyribonucleic acid synthesis, but protein synthesis continued at a diminished rate for a short time. Addition of chloramphenicol did not release RNA synthesis from inhibition at 40 C. Synthesis of beta-galactosidase could be induced at high temperature despite the presence of glucose in the medium, indicating a lesion in glucose catabolism. Of many catabolic enzymes tested in cell-free extracts, only fructose-1,6-diphosphate aldolase activity appeared to be altered in the mutant cells. No activity was demonstrable in extracts of mutant cells grown at either 30 or 40 C, but determination of glucose-oxidation patterns revealed that the enzyme is probably active in vivo at 30 C. Temperature-resistant secondary mutants were found to have partially or fully restored aldolase activity, and temperature-resistant recombinants had normal aldolase activity, indicating that the growth pattern and the altered aldolase had a common genetic basis. Linkage data permitted the assignment of an approximate map location for the mutated aldolase gene.     

Chloroplast and Cytoplasmic Enzymes: IV. Pea Leaf Fructose 1,6-Diphosphate Aldolases

Several peaks of aldolase activity are found in the isoelectric focusing pattern of pea (Pisum sativum) leaf chloroplast extracts. One peak, separated by 0.5 pH unit from the major chloroplast aldolase peak, is found when cytoplasmic extracts are focused. The chloroplast and cytoplasmic enzymes have a pH 7.4 optimum with fructose 1,6-diphosphate. The Michaelis constant for fructose-1,6-diphosphate is 19 μM for the chloroplast, 21 μM for the cytoplasmic enzyme, and for sedoheptulose 1,7-diphosphate, 8 μM for the chloroplast enzyme, 18 μM for the cytoplasmic enzyme. Both enzymes are inhibited by d-glyceraldehyde 3-phosphate and by ribulose 1,5-diphosphate. The similarity in the catalytic properties of the isoenzymes suggests that both enzymes have an amphibolic role in carbon metabolism in the green leaf.     

A new gene involved in expression of fructose-1,6-diphosphate aldolase activity in Escherichia coli.

A new gene, fdaB, has been mapped by transduction and partial diploid analyses and located adjacent to argA at 59.9 min on the Escherichia coli recalibrated linkage map. This gene is involved in expression of fructose-1,6-diphosphate aldolase activity and indirectly in ribosomal RNA synthesis. The temperature-sensitive mutant strain AA-157, containing the defective gene product of of fdaB, accumulates high concentrations of fructose 1,6-diphosphate at the nonpermissive temperature.     

The role of fructose-1, 6-diphosphate in cell migration and proliferation in an in vitro xenograft blood vessel model of vascular wound healing

Summary Both smooth muscle cells and endothelial cells play an important role in vascular wound healing. To elucidate the role of fructose-1, 6-diphosphate, cell proliferation and cell migration studies were performed with human endothelial cells and rat smooth muscle cells. To mimic blood vessels, endothelial and smooth muscle cells were used in 1:10, 1:5, and 1:1 concentrations, respectively, mimicking large-, mid-, and capillary-sized blood vessels. Cell migration was studied with fetal bovine serum-starved cells. For cell proliferation assay, cells were plated at 30–50% confluency and then starved. The cells were incubated for 48 h with fructose-1, 6-diphosphate at (per ml) 10 mg, 1 mg, 500 μg, 250 μg, 100 μg, and 10 μg, pulsed with tritiated-thymidine and incubated with 1 N NaOH for 30 min at room temperature, harvested, and counted. For migration assay, confluent cells were starved, wounded, and incubated for 24 h with same concentrations of fructose-1, 6-diphosphate as in proliferation assay. The cells were fixed and counted. Smooth muscle cell proliferation was inhibited by fructose-1, 6-diphosphate at 10 mg/ml. In the xenograft models of 1:10, 1:5, and 1:1 fructose-1, 6-diphosphate inhibited proliferation at 10 mg/ml. In migration studies 10 mg fructose-1, 6-diphosphate per ml was inhibitory to both cell types. In large-, mid-, and capillary-sized blood vessels, fructose-1, 6-diphosphate inhibited proliferation of both cell types at 10 mg/ml. At the individual cell level, fructose-1, 6-diphosphate is nonstimulatory to proliferation of endothelial cells while inhibiting migration, and it acts on smooth muscle cells by inhibiting both proliferation and migration.     

Modulation of the interaction between aldolase and glycerol-phosphate dehydrogenase by fructose phosphates

Kinetics of fructose-1,6-disphosphate aldolase (EC 4.1.2.13) catalyzed conversion of fructose phosphates was analyzed by coupling the aldolase reactions to the metabolically sequential enzyme, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), which interacts with aldolase. At low enzyme concentration poly(ethylene glycol) was added to promote complex formation of aldolase and glycerol-phosphate dehydrogenase resulting in a 3-fold increase in KM of fructose-1,6-bisphosphate and no change in Vmax. Kinetic parameters for fructose-1-phosphate conversion changed inversely upon complex formation: Vmax increased while KM remained unchanged. Gel penetration and ion-exchange chromatographic experiments showed positive modulation of the interaction of aldolase and dehydrogenase by fructose-1,6-bisphosphate. The dissociation constant of the heterologous enzyme complex decreased 10-fold in the presence of this substrate. Fructose-1-phosphate or dihydroxyacetone phosphate had no effect on the dissociation constant of the aldolase-dehydrogenase complex. In addition, titration of fluorescein-labelled glycerol-phosphate dehydrogenase with aldolase indicated that both fructose-1,6-bisphosphate and fructose-2,6-biphosphate enhanced the affinity of aldolase to glycerol-phosphate dehydrogenase. The results of the kinetic and binding experiments suggest that binding of the C-6 phosphate group of fructose-1,6-bisphosphate to aldolase complexed with dehydrogenase is sterically impeded while saturation of the C-6 phosphate group site increases the affinity of aldolase for dehydrogenase. The possible molecular mechanism of the fructose-1,6-bisphosphate modulated interaction is discussed.     

Properties of a Mutant of Escherichia coli with a Temperature-sensitive Fructose-1,6-Diphosphate Aldolase

B?ck, August (Purdue University, Lafayette, Ind.), and Frederick C. Neidhardt. Properties of a mutant of Escherichia coli with a temperature-sensitive fructose-1,6-diphosphate aldolase. J. Bacteriol. 92:470-476. 1966.-A mutant of Escherichia coli in which fructose-1,6-diphosphate aldolase functions at 30 C but not at 40 C was used to study the physiological effect of a specific block in the Embden-Meyerhof glycolytic pathway. Growth of the mutant at 40 C was found to be inhibited by the presence of glucose or certain related compounds in the medium. At 40 C, glucose was metabolized at 30 to 40% of the control rate and was abnormal in that glucose was converted into other six-carbon substances (probably gluconate, in large part) that were released into the culture medium. The inhibition was complete, but transient; its duration depended upon the initial amount of inhibitor added. The resumption of growth at 40 C was correlated with the further catabolism of the excreted compounds. When glycerol was used to grow the mutant at 40 C, the growth inhibition by glucose was accompanied by cessation of glycerol metabolism. Growth on alpha-glycerol phosphate was not inhibited under these conditions, implicating glycerol kinase as a possible site of inhibition; no inhibition of glycerol kinase by sugar phosphates, however, could be detected in vitro. The inhibitory effect of glucose on growth at 40 C is not caused by a deficit of intracellular adenosine triphosphate, but may be the result of a generalized poisoning of many cell processes by a greatly increased intracellular concentration of fructose-1,6-diphosphate, the substrate of the damaged enzyme.     

Histochemical method for demonstrating aldolase

Summary By combing the indirect method of aldolase activity of Warburg and Christian, which consisted in the measurement of reduction of DPN in the presence of glyceraldehyde-3-phosphate dehydrogenase and arsenate, with nitro-BT reduction and we could obtain the much better method of demonstrating aldolase than that of Allen and Bourne.The optimal incubating mixture was composed of 1) 10 ml 0.02 M sodium fructose-1,6-diphosphate, 2) 5 mg DPN, 3) 10 mg nitro-BT, 4) 10 ml of 0.05 M arsenate-HCl buffer (pH 7.6). Fresh frozen section, which were fixed briefly in 80% cold ethanol, gave a better staining results. The distribution of aldolase of some organs of rat and the validity and limitation of the method were described.     

Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.     

Metabolic alterations mediated by 2-ketobutyrate in Escherichia coli K12

Summary We have previously proposed that 2-ketobutyrate is an alarmone in Escherichia coli. Circumstantial evidence suggested that the target of 2-ketobutyrate was the phosphoenol pyruvate: glycose phosphotransferase system (PTS). We demonstrate here that the phosphorylated metabolites of the glycolytic pathway experience a dramatic downshift upon addition of 2-ketobutyrate (or its analogues). In particular, fructose-1,6-diphosphate, glucose-6-phosphate, fructose-6-phosphate and acetyl-CoA concentrations drop by a factor of 10, 3, 4, and 5 respectively. This result is consistent with (i) an inhibition of the PTS by 2-ketobutyrate, (ii) a control of metabolism by fructose-1,6-diphosphate. Since fructose-1,6-diphosphate is an activator of phosphoenol pyruvate carboxylase and of pyruvate kinase, the concentration of their common substrate, phosphoenol pyruvate, does not decrease in parallel.Abbreviations G1P glucose-1-phosphate - G6P glucose-6-phosphate - F6P fructose-6-phosphate - F1-6DP fructose-1,6-diphosphate - PEP phosphoenol pyruvate     

Inhibition of bovine hepatic fructose-1,6-diphosphatase by substrate analogs.

Purified bovine hepatic fructose-1,6-diphosphatase, which exhibits maximal activity at neutral pH, is competitively inhibited by several analogs of its substrate, fructose 1,6-diphosphate. These include glucose 1,6-diphosphate (Ki = 9.4 X 10(-5) M), hexitol 1,6-diphosphate (Ki = 2.3 X 10(-4) M), and 2,5-anhydro-D-mannitol 1,6-diphosphate (Ki = 3.3 X 10(-8) M), and 2,5-anhydro-D-glucitol 1,6-diphosphate (Ki = 5.5 X 10(-7) M). The Ki values for both 2,5-anhydro-D-mannitol 1,6-diphosphate and 2,5-anhydro-D-glucitol 1,6-diphosphate are lower than the Km of 1.4 X 10(-6) M for fructose 1,6-diphosphate. Since 2,5-anhydro-D-mannitol 1,6-diphosphate is an analog of the beta anomer of fructose 1,6-diphosphate and 2,5-anhydro-D-glucitol 1,6-diphosphate is an analog of the alpha anomer, the lower Ki for the mannitol analog may indicate that the beta anomer of fructose 1,6-diphosphate, which predominates in solution, is the true substrate. The substrate analog 1,5-pentanediol diphosphate inhibits slightly (K0.5 = 5 X 10(-3) M), but 1,4-cyclohexyldiol diphosphate does not. The Ki for product inhibition by sodium phosphate is 9.4 X 10(-3) M. 2,5-Anhydro-D-mannitol 1,6-diphosphate and alpha-D-glucose 1,6-diphosphate are substrates at pH 9.0, but not at pH 6.5.     

The Escherichia coli ts8 mutation is an allele of fda, the gene encoding fructose-1,6-diphosphate aldolase.

The ts8 mutant of Escherichia coli has previously been shown to preferentially inhibit stable RNA synthesis when shifted to the nonpermissive temperature. We demonstrate in this report that the ts8 mutation is an allele of fda, the gene that encodes the glycolytic enzyme fructose-1,6-diphosphate aldolase. We show that ts8 and a second fda mutation, h8, isolated and characterized by A. B?ck and F. C. Neidhardt, are dominant mutations and that they encode a thermolabile aldolase activity.     

Two Class I Aldolases in the Green Alga Chara foetida (Charophyceae)

Aldolase activity of Chara foetida (Braun) could be separated into a minor (peak I) and a major peak (peak II) by ion-exchange chromatography on DEAE-cellulose. Affinity chromatography on P-cellulose resulted in highly purified aldolase preparations with specific activities of 3.2 and 4.8 units per milligram protein and molecular subunit masses of 37 and 35 kilodalton, as shown by SDS-PAGE, for the aldolase of peak I and peak II, respectively. Both aldolases belong to class I aldolase since the activity is not inhibited by 1 millimolar EDTA. The K_m (fructose-1,6-bisphosphate) values were 0.64 and 13.4 micromolar, respectively. The aldolase of peak I showed a 6.7 times stronger crossreaction with a specific antiserum against the cytosol aldolase of spinach than with an antiserum against the chloroplast aldolase of spinach. On the other hand the aldolase of peak II showed a 5.1 times stronger cross-reaction with the α-plastidaldolase antiserum than with the α-cytosol-aldolase antiserum. For algae this is the first separation of two class I aldolases. They are similar to the cytosol and chloroplast aldolases in higher plants, but different from a reported class I (Me²⁺ independent) and class II (Me²⁺ dependent) aldolase in other algae.     

Modification of human erythrocyte pyruvate kinase by an active site-directed reagent: bromopyruvate

Human erythrocyte pyruvate kinase was modified with bromopyruvate and the kinetic behavior of the modified enzyme was investigated. When the enzyme was modified with bromopyruvate in the absence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate the inactivation followed a pseudo first-order kinetics. The inactivation rate constant, ks, was 1.84 +/- 0.15 min(-1). Kd of the bromopyruvate-enzyme complex was 0.14 +/- 0.03 mM. The presence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate in the modification medium or the presence of fructose-1,6-diphosphate in the assay medium resulted in deviation of the inactivation kinetics from pseudo first-order. Phosphoenolpyruvate was better than adenosine-5'-diphosphate for protection against bromopyruvate modification whereas fructose-1,6-diphosphate was ineffective. The modified enzyme showed negative cooperativity in the presence of fructose-1,6-diphosphate whereas in the absence of it no activity was detected.     

Modification of Human Erythrocyte Pyruvate Kinase by an Active Site-directed Reagent: Bromopyruvate

Human erythrocyte pyruvate kinase was modified with bromopyruvate and the kinetic behavior of the modified enzyme was investigated. When the enzyme was modified with bromopyruvate in the absence of adenosine-5′s-diphosphate, phospho-enolpyruvate or fructose-1,6-diphosphate the inactivation followed a pseudo first-order kinetics. The inactivation rate constant, k_s, was 1.84 × 0.15 min^?1. K_d of the bromopyruvate-enzyme complex was 0.14 × 0.03 mM.

The presence of adenosine-5′-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate in the modification medium or the presence of fructose-1,6-diphosphate in the assay medium resulted in deviation of the inactivation kinetics from pseudo first-order. Phosphoenolpyruvate was better than adenosine-5′-diphosphate for protection against bromopyruvate modification whereas fructose-1,6-diphosphate was ineffective. The modified enzyme showed negative cooperativity in the presence of fructose-1,6-diphosphate whereas in the absence of it no activity was detected.