Legionella pneumophila Is Directly Sensitive to 2-Deoxyglucose-Phosphate via Its UhpC Transporter but Is Indifferent to Shifts in Host Cell Glycolytic Metabolism.

Toll-like receptor (TLR) stimulation induces a pronounced shift to increased glycolytic metabolism in mammalian macrophages. We observed that bone marrow-derived macrophages (BMMs) increase glycolysis in response to infection with Legionella pneumophila, but the role of host macrophage glycolysis in terms of intracellular L. pneumophila replication is not currently understood. Treatment with 2-deoxyglucose (2DG) blocks L. pneumophila replication in mammalian macrophages but has no effect on bacteria grown in broth. In addition, we found that 2DG had no effect on bacteria grown in amoebae. We used a serial enrichment strategy to reveal that the effect of 2DG on L. pneumophila in macrophages requires the L. pneumophila hexose-phosphate transporter UhpC. Experiments with UhpC-deficient L. pneumophila revealed that mutant bacteria are also resistant to growth inhibition following treatment with phosphorylated 2DG in broth, suggesting that the inhibitory effect of 2DG on L. pneumophila in mammalian cells requires 2DG phosphorylation. UhpC-deficient L. pneumophila replicates without a growth defect in BMMs and protozoan host cells and also replicates without a growth defect in BMMs treated with 2DG. Our data indicate that neither TLR signaling-dependent increased macrophage glycolysis nor inhibition of macrophage glycolysis has a substantial effect on intracellular L. pneumophila replication. These results are consistent with the view that L. pneumophila can employ diverse metabolic strategies to exploit its host cells.IMPORTANCE We explored the relationship between macrophage glycolysis and replication of an intracellular bacterial pathogen, Legionella pneumophila Previous studies demonstrated that a glycolysis inhibitor, 2-deoxyglucose (2DG), blocks replication of L. pneumophila during infection of macrophages, leading to speculation that L. pneumophila may exploit macrophage glycolysis. We isolated L. pneumophila mutants resistant to the inhibitory effect of 2DG in macrophages, identifying a L. pneumophila hexose-phosphate transporter, UhpC, that is required for bacterial sensitivity to 2DG during infection. Our results reveal how a bacterial transporter mediates the direct antimicrobial effect of a toxic metabolite. Moreover, our results indicate that neither induction nor impairment of host glycolysis inhibits intracellular replication of L. pneumophila, which is consistent with a view of L. pneumophila as a metabolic generalist.

Inhibition of Recombinant Aldose-6-Phosphate Reductase from Peach Leaves by Hexose-Phosphates, Inorganic Phosphate and Oxidants.

Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.

Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature.

Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-¹4C]glucose 1-phosphate, [U-¹4C]sucrose, [U-¹4C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-¹4C]sucrose plus unlabelled equimolar glucose 1-phosphate. C¹4-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced ¹4C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-¹4C]glucose 1-phosphate or adenosine-[U-¹4C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C¹4C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.

Glucose and its metabolites have distinct effects on the calcium-induced mitochondrial permeability transition.

Mitochondrial production of reactive oxygen species (ROS) due to up-regulated glucose oxidation is thought to play a crucial, unifying role in the pathogenesis of chronic complications associated with diabetes mellitus. Mitochondrial permeability transition (MPT) is an interesting phenomenon involved in calcium signalling and cell death. We investigated the effects of glucose and several of its metabolites on calcium-induced MPT (measured as mitochondrial swelling) in isolated rat liver mitochondria. The presence of glucose, glucose 1-phosphate (both at 30 mM) or methylglyoxal (6 mM) significantly slowed calcium-induced mitochondrial swelling. Thirty mM glucose also resulted in a significant delay of MPT onset. In contrast, 30 mM fructose 6-phosphate accelerated swelling, whereas glucose 6-phosphate did not influence the MPT. The calcium binding potentials of the three hexose phosphates were tested and found similar. In vitro hydrogen peroxide production by mitochondria respiring on succinate in the presence of rotenone was independent of mitochondrial membrane potential and increased transiently during calcium-induced MPT. Inhibition of MPT with cyclosporine A resulted in decreased mitochondrial ROS production in response to calcium. In contrast, inhibition of MPT by methylglyoxal was accompanied by increased ROS production in response to calcium. In conclusion, we confirm that methylglyoxal is a potent inhibitor of MPT. In addition, high levels of glucose, glucose 1-phosphate and fructose 6-phosphate can also affect MPT. Methylglyoxal simultaneously inhibits MPT and increases mitochondrial ROS production in response to calcium. Our findings provide a novel context for the role of MPT in glucose sensing and the cellular toxicity caused by methylglyoxal.

A protein phosphorylation switch at the conserved allosteric site in GP.

A phosphorylation-initiated mechanism of local protein refolding activates yeast glycogen phosphorylase (GP). Refolding of the phosphorylated amino-terminus was shown to create a hydrophobic cluster that wedges into the subunit interface of the enzyme to trigger activation. The phosphorylated threonine is buried in the allosteric site. The mechanism implicates glucose 6-phosphate, the allosteric inhibitor, in facilitating dephosphorylation by dislodging the buried covalent phosphate through binding competition. Thus, protein phosphorylation-dephosphorylation may also be controlled through regulation of the accessibility of the phosphorylation site to kinases and phosphatases. In mammalian glycogen phosphorylase, phosphorylation occurs at a distinct locus. The corresponding allosteric site binds a ligand activator, adenosine monophosphate, which triggers activation by a mechanism analogous to that of phosphorylation in the yeast enzyme.

Human erythrocyte hexokinase deficiency. Characterization of a mutant enzyme with abnormal regulatory properties.

In the erythrocytes of a patient with hereditary nonspherocytic hemolytic anemia, a homozygous expression of hexokinase deficiency was detected. The mutant enzyme was characterized by normal kinetic parameters with respect to its substrates, glucose and MgATP2-, normal pH optimum, normal heat stability at 40 degrees C, but abnormal behavior with respect to its regulation by glucose-1,6-diphosphate and inorganic phosphate, and an altered electrophoretic pattern. Interpretation of the results revealed the presence of two different hexokinases type I in normal human erythrocytes: one enzyme with a high affinity for glucose-1,6-diphosphate, the inhibition of which is regulated by inorganic phosphate; and another enzyme with a lower affinity for the inhibitor, not regulated by inorganic phosphate. The former enzyme was not detectable in the erythrocytes of the patient, whereas the presence of the latter enzyme could be demonstrated.

Anomeric specificity of hexokinase in rat, human, and murine tumor cells.

In tumoral cells derived from the insulin-producing rat cell line RINm5F, both low- and high-Km glucose-phosphorylating enzymic activities were present. The hexokinase-like enzyme was inhibited by glucose 6-phosphate and displayed a greater affinity for but lower maximal velocity with alpha-D-glucose than beta-D-glucose. A comparable anomeric behavior of hexokinase was observed in breast cancer (MCF-7) and lymphocytic leukemia (P388) cells. Thus, the anomeric specificity of hexokinase in tumoral cells was not different from that recently characterized in normal mammalian cells.

Inhibition of potato starch phosphorylase by alpha-D-glucopyranose-1,2-cyclic phosphate.

alpha-D-Glucopyranose-1.2-cyclic phosphate is a potent inhibitor of potato starch phosphorylase-catalyzed (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) starch elongation. The inhibition is competitive with respect to alpha-D-glucopyranose 1-phosphate (Glc-1-P) with Ki approximately 0.07 mM at pH 6.3 and 30 degrees C in 25 mM citrate buffer. The affinity of the phosphorylase - starch complex for the cyclic ester is therefore nearly 30 times as large as for Glc-1-P. Under conditions where alpha-D-glucopyranose-1,2-cyclic phosphate slows starch elongation by a factor of 3, UDPglucose, ADPglucose, D-glucose 6-phosphate, and D-glucose 2-phosphate cause rate reductions of less than 10%. The origin of the relatively strong binding of the cyclic ester to the phosphorylase, and its possible biological significance are discussed.

Kinetic analysis of effectors of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi.

The activity of phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxy-lyase (phosporylating) EC 4.1.1.31) purified from Bryophyllum fedtschenkoi has been measured in the presence of various concentrations of phosphoenolpyruvate, L-malate and glucose 6-phosphate. At high pH, the enzyme is competitively inhibited by L-malate and activated by glucose 6-phosphate. A reaction scheme describing the interaction of enzyme, substrate and effectors is proposed. Values for the appropriate equilibrium constants have been calculated for the enzyme acting at pH 7.8, which is one of its two pH optima. The kinetics are more complicated at low pH, partly because of non-linear reaction rates and partly because inhibition by L-malate is not competitive. Activation by glucose 6-phosphate is similar at high and low pH values. The behaviour of a wide range of other possible effectors is described briefly.

Hexokinase isozyme distribution and regulatory properties in lymphoid cells.

The glycolytic enzyme hexokinase is studied in cultured leukemic lymphoblasts, in normal lymphocytes and in lymphoblasts obtained by stimulation of normal lymphocytes with phytohaemagglutinin. Hexokinase activity levels in cultured lymphoblasts and in normal lymphocytes are identical, but somewhat higher levels are found in stimulated lymphocytes. Cultured leukemic lymphoblasts differ in isozyme content in comparison to the other lymphoid cells. Besides hexokinase I, which is detected in all the lymphoid cells, they are characterized by the presence of hexokinase II. The concentration of type II increases during cell growth. Another difference between leukemic lymphoblasts and mature and stimulated lymphocytes is found in the regulatory properties of hexokinase I. Hexokinase I from both normal and stimulated lymphocytes is inhibited by glucose-1,6-diphosphate. This inhibition is decreased in part by addition of inorganic phosphate. Hexokinase I from leukemic lymphocytes, however, is inhibited to a lesser extent by glucose-1,6-diphosphate. Inorganic phosphate has no effect at all on this inhibition. In accordance with these findings a different pattern in the hexokinase I region was detected in electrophoresis with several cell types. The subisozyme hexokinase Ib, which appears to be the phosphate-regulated form, is predominant in lymphocytes, whereas it is present in a minor fraction in the cultured leukemic lymphoblasts. In these cells hexokinase Ic predominates.

Inhibition of 6-phosphogluconate dehydrogenase (decarboxylating) by glucose 1,6-bisphosphate.

Glucose 1,6-bisphosphate, the powerful common regulator of several key enzymes in carbohydrate metabolism, was found to exert a potent inhibitory effect on the activity of 6-phosphogluconate dehydrogenase (decarboxylating) from yeast and several rat tissues. These findings suggest that glucose 1,6-bisphosphate may have a regulatory influence on the pentose phosphate pathway.

Inhibition of phosphoglucomutase by fructose 2,6-bisphosphate.

Fructose 2,6-bisphosphate inhibits phosphoglucomutase. The inhibition is mixed with respect to glucose 1,6-bisphosphate and non-competitive with respect to glucose 1-phosphate. In contrast with fructose 1,6-bisphosphate and glycerate 1,3-bisphosphate, which also possess inhibitory effect, fructose 2,6-bisphosphate does not phosphorylate phosphoglucomutase. Fructose 2,6-bisphosphate preparations contain contaminants which can explain artefactual results previously reported.

Comparison of type I hexokinases from pig heart and kinetic evaluation of the effects of inhibitors.

Type I hexokinase (ATP:D-hexose 6-phospotransferase, EC 2.7.1.1) of porcine heart exists in two chromatographically distinct forms. These do not differ significantly in size, electrophoretic mobility at pH 8.6 or kinetic properties. Both forms obey a sequential mechanism and are potently inhibited by glucose 6-phosphate. In contrast to observations of type I hexokinase from brain, inhibition by glucose 6-phosphate is not relieved by inorganic phosphate. Under most conditions, low concentrations of phosphate (less than 10 mM) have little effect on the kinetic behaviour of the enzyme but at higher concentrations this ligand is an inhibitor. Mannose 6-phosphate inhibits in a manner analogous to glucose 6-phosphate but the Ki is much greater. In view of the similarity of the kinetic parameters governing phosphorylation of mannose and glucose, this difference in affinity for the inhibitor site is seen as consistent with the existence of a separate regulatory site on the enzyme. MgADP inhibits hexokinase but behaves as a normal product inhibitor and inhibition is competitive with respect to MgATP and non-competitive with respect to glucose.

The preparation of transketolase free from D-ribulose-5-phosphate 3-epimerase.

A procedure for the purification from Candida utilis of transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) free from D-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) was developed using acetone precipitation, elution from DEAE-cellulose, adsorption of epimerase by thiopropyl-Sepharose, and chromatography on D-ribose 5-phosphate-Sepharose and DEAE--Sephadex. The final product had a specific activity of 43 units/mg, a transketolase/epimerase activity ratio greater than 53 000 to 1, an apparent Km for D-xylulose 5-phosphate and D-ribose 5-phosphate of 77 and 430 microM, respectively, and ran as a single band using electrophoresis on polyacrylamide gel. It was inhibited by D-arabinose 5-phosphate and D-glucose 6-phosphate. During the purification by column chromatography, multiple forms of the enzyme were detected by gel electrophoresis but these gradually disappeared as the enzyme was further purified.

On the mechanism of inactivation of muscle glycogen phosphorylase by insulin.

Glucose 6-phosphate, an allosteric inhibitor of skeletal muscle phosphorylase b, inhibits at physiological concentrations and conditions the phosphorylation and activation of the enzyme by phosphorylase b kinase. AMP inhibits the dephosphorylation of phosphorylase a, but is without effect on the phosphorylation of phosphorylase b. Glucose 6-phosphate has no effect on the activity of phosphorylase a and does not affect its dephosphorylation by phosphatases 1 or 2A. The inhibition of the phosphorylation of phosphorylase b by glucose 6-phosphate may explain the reported decreased phosphorylation of phosphorylase in muscle following insulin treatment, which elevates intracellular levels of glucose 6-phosphate.

Glucose 6-phosphate-dependent binding of hexokinase to membranes of ascites tumor cells.

A pH-dependent, saturable binding of hexokinase isozyme I from Ehrlich ascites carcinoma to plasma membrane and microsome preparations from the same tissue is demonstrated. This binding is enhanced by glucose 6-phosphate and may be considered as the sum of a glucose 6-phosphate-dependent binding and an independent binding. The half saturation concentration of hexokinase is about 0.4 unit per ml for both types of binding, and a maximal binding of 0.5-2.0 units per mg membrane protein is observed for both, although the pH optimum of the independent binding (5.4) is lower than that of the dependent binding (5.9). The half saturation concentration of glucose 6-phosphate required for the dependent binding is 0.05 mM at pH 6.1. 2-Deoxyglucose 6-phosphate competatively reverses the effect of glucose 6-phosphate on binding but does not diminish its inhibition of hexokinase activity.

Reversible microsomal binding of hepatic aldolase.

Fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) partitions between the microsomes and the cytosol when a rat liver homogenate is fractionated by differential centrifugation. Gel electrophoresis and immunodiffusion indicate that the one isozyme present in the liver of the young adult rat is found in both fractions. The association of the aldolase with membranes is differentially sensitive to a variety of metabolites and inorganic salts. In the absence of cellular salts, 1 mM fructose 1,6-bisphosphate or glucose 1,6-bisphosphate elutes 50% of the enzyme from the microsomes. About 9 mM Pi or citrate is necessary to produce the same effect. With other metabolites or inorganic salts higher concentrations are required. The fraction of total enzyme which partitions with the microsomes when a homogenate is submitted to high speed centrifugation, correlates inversely with the level of fructose 1,6-bisphosphate in the supernatant solution and this concentration is higher when the tissue concentration in the homogenate is greater. The Km for fructose 1,6-bisphosphate of 3 . 10(-4) for aldolase bound to microsomes is decreased to 6 . 10(-6) M when the enzyme is dissociated from the membranes with salt. These observations appear relevant to the ongoing discussion regarding the physiological relevance of the subcellular localization of glycolytic enzymes.

Location of the aromatic binding site and preparation of an aromatic derivative of glycogen phosphorylase.

Rabbit muscle glycogen phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) was inactivated by 1,5-difluoro-2,4-dinitrobenzene at pH 7.6 at a rate much faster than by 1-fluoro-2,4-dinitrobenzene. The reaction was very specific at low concentration of 1,5-difluoro-2,4-dinitrobenzene. Glucose 1-phosphate, glucose 6-phosphate, AMP and ATP afforded some protection against inactivation by 1,5-difluoro-2,4-dinitrobenzene. These results and kinetic analyses of the modified enzyme were used to locate the binding site for aromatic compounds in phosphorylase. The above ligands and aromatic compounds are shown to bind on the enzyme in the same region which is located near the monomer/monomer interface. An apparently homogeneous dinitrodiphenyzene derivative of phosphorylase b with only one group per dimeric enzyme and having 50% of the catalytic activity was prepared. This derivative in which the subunits were not cross-linked by the reagent was devoid of the homotropic cooperativity for the substrate or activator sites even in the presence of allosteric inhibitors. Glucose behaved quite differently from other ligands in its effect on modification and on the kinetics of the modified enzyme. The significance of the glucose site is discussed.

Separation and characterization of hexokinase I subtypes from human erythrocytes.

Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) type 1 from human erythrocytes exists in four electrophoretical distinct forms, termed Ia, Ib, Ic and Id in order of their increasing anodal electrophoretic mobility at pH 8.8. We were able to separate type Ia, Ib and Icd on phosphocellulose by using a discontinuous gradient elution. The three chromatographically distinct forms do not differ in their affinity constants for the substrates glucose and MgATP2-. In addition the inhibition by glucose 1,6-diphosphate does not differ significantly for all forms. However, the regulation of these inhibitions by inorganic phosphate is much less for type Ia compared to the other subtypes (P = 0.001). Aging of the red cells is accompanied by a relative increase of the proportion of type Ic and Ia, which is the less regulated form of the enzyme. This shift in electrophoretic and regulatory properties is argued to be due to a post-translational modification of the primary enzyme.

Digestive end products release pancreatic enzymes from particulate cellular pools, particularly zymogen granules.

The effects of various amino acids and phosphorylated forms of glucose on the release of digestive enzymes from particulate cellular pools, particularly zymogen granules, were evaluated in rat pancreas. Whole tissue homogenates, as well as zymogen granules isolated either by differential centrifugation in 0.3 M sucrose or by preparation in buffered sucrose and subsequent centrifugation in a Percoll gradient, were studied. The basic amino acids L-arginine and L-lysine, sites of tryptic cleavage, caused the release of trypsinogen, but not chymotrypsinogen, whereas the aromatic amino acids L-phenylalanine and L-tryptophan, sites of chymotryptic cleavage, caused release of both trypsinogen and chymotrypsinogen. Neither led to the release of the starch-splitting enzyme amylase. All effects occurred within the range of normal plasma concentrations for these amino acids in the rat. Two amino acids, L-threonine and hydroxy-L-proline, that are not sites of cleavage by trypsin or chymotrypsin, and a nonmammalian amino acid, aminoadipic acid, did not lead to release of trypsinogen, chymotrypsinogen, or amylase. Two phosphorylated forms of glucose, glucose 1-phosphate and glucose 1,6-diphosphate, caused the release of amylase, but of neither trypsinogen nor chymotrypsinogen. Contrary to previous results, D-glucose was without effect, as was glucose 6-phosphate. We propose that certain digestive end products, by direct action on zymogen granules, cause the selective release of the enzymes involved in their evolution from polymeric substrates during digestion.