Inhibition of hexose transport by glucose in a glucose-6-phosphate isomerase mutant ofSaccharomyces cerevisiae

The rate of hexose transport was approximately 60% lower for both the high- and the low-affinity components of hexose uptake when a glucose-6-phosphate isomerase mutant ofSaccharomyces cerevisiae was preincubated with glucose, as compared with preincubation with water. Similarly theJ _max value of the high-affinity system of the mutant was 25–35 % of the correspondingJ _max value for normal cells incubated with glucose. Accumulation of glucose 6-phosphate or of some other metabolite, such as fructose 6-phosphate or trehalose, may be responsible for this striking inhibition.     

Biosynthetic pathway of sugar nucleotides essential for welan gum production in Alcaligenes sp. CGMCC2428

Welan gum is a microbial polysaccharide produced by Alcaligenes sp. CGMCC2428 that has d-glucose, d-glucuronic acid, d-glucose, and l-rhamnose as the main structural unit. The biosynthetic pathway of sugar nucleotides essential for producing welan gum in this strain was established in the following ways: (1) the detection of the presence of several intermediates and key enzymes; (2) the analysis of the response upon addition of precursors to the culture medium; (3) the correlation of the activities between several key enzymes with the yields of welan gum. With addition of 200-μM glucose-6-phosphate and fructose-6-phosphate, the production of welan gum was improved by 18%. The activities of phosphoglucomutase, phosphomannose isomerase, UDP-glucose pyrophosphorylase, and dTDP-glucose pyrophosphorylase, correlated well with the yields of welan gum. According to these findings, the biosynthetic pathway was proposed to involve the metabolism of glucose via two discrete systems. The first involves conversion of glucose to glucose-6-phosphate, with further reactions producing glucose-1-phosphate and fructose-6-phosphate, which are metabolized to the nucleotide sugar precursors of welan gum. The second system involves metabolism of glucose to synthesize the basic structural skeleton of the cell via central metabolic pathways, including the Entner–Doudoroff pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle.     

Identification and functional reconstitution of phosphate: Sugar phosphate antiport ofStaphylococcus aureus

Summary Resting cells ofStaphylococcus aureus displayed a phosphate (P_i) exchange that was induced by growth with glucose 6-phosphate (G6P) orsn-glycerol 3-phosphate (G3P). P_i-loaded membrane vesicles from these cells accumulated³²P_i, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of³²P_i (andl-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (dl-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both P_i anddl-lactate to establish an internal pool of P_i. Thistrans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal P_i.Reconstitution of membrane protein allowed a quantitative analysis of P_i-linked exchange. P_i-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous³²P_iP_i exchange (K _i's of 2.2 and 1.4mm;V _max's of 180 and 83 nmol P_i/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (K _i=27 m) over G3P (K _i=1.3mm) and P_i (K _i=2.2mm), suggesting that the same antiporter was induced in both cases. We conclude that³²P_iP_i exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate. In this respect, P_i-linked antiport inS. aureus resembles other examples in a newly described family of bacterial transporters that use anion exchange as the molecular basis of solute transport.     

Metabolic regulation of the glucose-6-phosphate dehydrogenase from Paracoccus denitrificans grown on glucose/nitrate

Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP⁺ l-oxidoreductase EC 1.1.1.49) isolated from Paracoccus denitrificans grown on glucose/nitrate exhibits both NAD⁺-and NADP⁺-linked activities. Both activities have a pH optimum of pH 9.6 (Glycine/NaOH buffer) and neither demonstrates a Mg²⁺ requirement. Kinetics for both NAD(P)⁺ and glucose-6-phosphate were investigated. Phosphoenolpyruvate inhibits both activities in a competitive manner with respect to glucose-6-phosphate. ATP inhibits the NAD⁺-linked activity competitively with respect to glucose-6-phosphate but has no effect on the NADP⁺-linked activity. Neither of the two activities are inhibited by 100 M NADH but both are inhibited by NADPH. The NAD⁺-linked activity is far more sensitive to inhibition by NADPH than the NADP⁺-linked activity.     

Substrate specificity of a glucose-6-phosphate isomerase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> for monosaccharides

We purified recombinant glucose-6-phosphate isomerase from Pyrococcus furiosus using heat treatment and Hi-Trap anion-exchange chromatography with a final specific activity of 0.39 U mg⁻¹. The activity of the glucose-6-phosphate isomerase for l-talose isomerization was optimal at pH 7.0, 95°C, and 1.5 mM Co²⁺. The half-lives of the enzyme at 65°C, 75°C, 85°C, and 95°C were 170, 41, 19, and 7.9 h, respectively. Glucose-6-phosphate isomerase catalyzed the interconversion between two different aldoses and ketose for all pentoses and hexoses via two isomerization reactions. This enzyme has a unique activity order as follows: aldose substrates with hydroxyl groups oriented in the same direction at C2, C3, and C4 > C2 and C4 > C2 and C3 > C3 and C4. l-Talose and d-ribulose exhibited the most preferred substrates among the aldoses and ketoses, respectively. l-Talose was converted to l-tagatose and l-galactose by glucose-6-phosphate isomerase with 80% and 5% conversion yields after about 420 min, respectively, whereas d-ribulose was converted to d-ribose and d-arabinose with 53% and 8% conversion yields after about 240 min, respectively.     

Isoenzyme evidence on the systematics ofHordeum sectionMarina (Poaceae)

Morphological differentiation of diploid accessions ofHordeum marinum Huds. s.l. into two varieties, var.marinum and var.fouilladei (Rouy)Nevski is associated with isoenzyme differentiation. The tetraploid form ofH. marinum s.l. exhibited fixed heterozygosity of several heterozymes with one homoeozyme shared with var.fouilladei and the second homoeozyme not found in the two diploids. It also differed from both diploids in the mobility of glucose-6-phosphate dehydrogenase. All three taxa differed in morphs of EST-A. It is concluded that the tetraploid is an allopolyploid with one genome closely related to the diploid var.fouilladei and with the second genome divergent from those of both diploids by genes for unique morphs of eight homoeozymes. On the basis of the isoenzyme data, three phylogenetic sibling species—H. marinum Huds. s.str. (2x),H. geniculatum All. s.str. (= var.fouilladei, 2x), andH. caudatum Jaaska, spec. nova (4x), are proposed within theH. marinum s.l. complex and a key is given.     

Energy coupling of the hexose phosphate transport system in Escherichia coli

The active transport of hexose phosphates in Escherichia coli was inhibited by many uncouplers or inhibitors of oxidative metabolism. Fluoride and the lipid soluble cation, triphenylmethylphosphonium, had little effect. The uninduced level of transport was sensitive to fluoride, but not to azide. After energy uncoupling of active transport, the cells could equilibrate their intracellular water with the glucose-6-phosphate in the medium and displayed exit counter-flow suggesting the existence of carrier-mediated transport in the energy-uncoupled cells. The uncoupled transport of glucose-6-phosphate was inhibited by fructose-6-phosphate; the uninduced level of glucose-6-phosphate transport was not inhibited by fructose-6-phosphate. After energy uncoupling, the influx had a low affinity suggesting that, unlike the transport of beta-galactosides, the energy coupling for the active transport of hexose phosphate involved a change in the affinity of influx.     

The effect of an anionic detergent on complex carbohydrates and enzyme activities in the epidermis of the catfish Heteropneustes fossilis (Bloch)

Summary The histochemistry of various oxidative enzymes and complex carbohydrates in the epidermis of the catfishHeteropneustes fossilis was investigated after exposure to sublethal concentrations of the detergent sodium alkulbenzenesulphonate.It was found that the detergent treatment was accompanied by a marked increase in the number of mucous cells which produce histochemically detectable amounts of acidic glycoproteins with a shift towards the production ofO-acetylated sialic acids. The activities of mitochondrial enzymes were lost in the superficial cell layers. In contrast the activities of glucose-6-phosphate and lactate dehydrogenase increased considerably. The rise in glucose-6-phosphate dehydrogenase was correlated with the metabolic requirements for the enhanced production of mucus under stress.The changes in both enzyme activities and in the chemical composition of mucus may provide a suitable experimental model for histochemical investigations of the effect of stress induced by pollulants on aquatic organisms.     

Phytohemagglutinin-enhanced hybrid colony formation by cells from aged mice: Expression of th 4mod-1 mouse locus in human-mouse hybrids

Summary Cells from a continuous human line and freshly isolated cells from old adult mice heterozygous at theMod-1 locus were fused in the presence of polyethylene glycol (PEG). The production of hybrid cells, as a function of PEG concentration in the presence and absence of phytohemagglutining (PHA), was measured by cell survival and proliferation on selective medium. The incorporation of PHA into the fusion mixture allowed cell fusion to take place at nontoxic concentrations of PEG. PHA increased the frequency of cell fusion and increased the production of viable hybrid cells from 138- to over 2800-fold depending on cell type. The results suggest that the procedure may have broad application in promoting the fusion of cells sensitive to PEG. Clones were analyzed for isozymes of malic enzyme and glucose-6-phosphate dehydrogenase. The expression of the gene encoding X-linked mouse glucose-6-phosphate dehydrogenase confirmed that the cells were hybrids. These cells lost other mouse isozymes rapidly. In those clones in which the mouse malic enzyme gene was expressed, the product ofMod-1 ^α was detected significantly more frequently than that ofMod-1 ^b.     

Active transport of glucose-1-phosphate in Agrobacterium tumefaciens

The presence of an active transport system for glucose-1-phosphate in Agrobacterium tumefaciens was demonstrated from the following observations. (i) The bacterium could grow on a medium containing glucose-1-phosphate as carbon source; (ii) the entry of glucose-1-phosphate into the resting cells occurred against concentration gradient obeying Michaelis-Menten kinetics; and (iii) the entry reaction was energy-dependent. The transport system for glucose-1-phosphate was formed inducibly by growing the organism on a glucose-1-phosphate or sucrose medium. From the inhibition and kinetics studies it was found that the transport system had a high specificity for glucose-1-phosphate with a high affinity, K(m) value of 4.5 x 10(-6)m at pH 8.2. The existence of glucose-1-phosphate binding factor was proved in the shock fluid which was prepared from the cells grown on both glucose-1-phosphate and sucrose media by osmotic shock.     

Isolation and regeneration of protoplasts from gills ofAgaricus bisporus

Protoplasts were isolated from fruit-bodies ofAgaricus bisporus, and highest yields were derived from basidia. When gill fragments were treated with a combination of Novozyme 234, chitinase, and cellulase Onozuka R-10, and with 0.35m KCl as the osmotic stabilizer, high yields (3–4×10⁷ protoplasts/g fresh wt gills) were obtained within 1 h of incubation. About 20–30% of protoplasts regenerated in a solid MMNC medium. Investigation of specific activities of glucose-6-phosphate dehydrogenase and mannitol dehydrogenase indicated a highly active pentose phosphate pathway and a good capacity for mannitol synthesis in protoplasts, as well as in other cells of fruit-bodies of the species. The simple and efficient procedure provides a new approach for further investigation of the mushroom, and possibly of other basidiomycetes with hemiangiocarpus, by use of protoplasts.     

Physiological studies on Phymatotrichum omnivorum III. Enzymes of glucose catabolism

Specific enzymes of Embden-Meyerhof-Parnas pathway and hexose-monophosphate pathway were detected in cell-free extract ofPhymatotrichum omnivorum grown on a synthetic medium. Cell-free extracts contained active phosphoglucoisomerase, glucose-6-phosphate and 6-phosphogluconate dehydrogenases of the EMP and HMP respectively. Phosphoglucomutase activity could not be detected in enzyme preparations.     

Transient repression of beta-galactosidase synthesis by glucose-6-phosphate in a mutant of Escherichia coli lacking enzyme II specific for glucose in the phosphoenolpyruvate-sugar phosphotransferase system.

The effects of glucose and glucose-6-phosphate in initiating the repression of beta-galactosidase synthesis were studied using a mutant of Escherichia coli K12 which lacks glucose-specific enzyme II of the phosphoenolpyruvate-sugar phosphotransferase system. It was found that glucose-6-phosphate causes transient repression of beta-galactosidase synthesis but glucose does not cause transient repression in this mutant. Evidence was obtained that both the presence of an active transport system for glucose-6-phosphate in the cells and glucose-6-phosphate in the medium are necessary for the initiation of transient repression. No metabolism of glucose-6-phosphate is required. Upon depletion of glucose-6-phosphate in the medium the transient repression was reversed. After the reversal the rate of enzyme synthesis was high in the cells which had been exposed to a high concentration of glucose-6-phosphate. It was concluded that the translocation of glucose-6-phosphate across the membranes is the primary event which affects both the initiation of and the recovery from the transient repression. During the transient repression the cellular content of cyclic adenosine 3',5'-monophosphate decreased significantly.     

Studies on the metabolism ofAcetobacter peroxydans

Summary Some physiological and biochemical properties of several strains ofAcetobacter peroxydans have been studied. Their morphology, the aspects of growth on beer-gelatine slants and the colony type are described. The cells are catalase negative and acid resistant. The temperature optimum is 20–25°C. They grow readily on ethanol and on lactate, or on yeast extract alone, but not on carbohydrates and derivatives. They do not consume or oxidize glucose or gluconate. They are overoxidizers. None of these strains was able to grow as hydrogen bacteria. Resting cells oxidize lactate, pyruvate, ethanol, acetate, some Krebs cycle intermediates and several alcohols. Cell-free extracts oxidize glucose-6-phosphate, 6-phosphogluconate and ribose-5-phosphate in suitable conditions. These results favour the taxonomic position of this species as a member of the genusAcetobacter. The results are discussed.     

Regulation of alternate peripheral pathways of glucose catabolism during aerobic and anaerobic growth of Pseudomonas aeruginosa.

Glucose may be converted to 6-phosphogluconate by alternate pathways in Pseudomonas aeruginosa. Glucose is phosphorylated to glucose-6-phosphate, which is oxidized to 6-phosphogluconate during anaerobic growth when nitrate is used as respiratory electron acceptor. Mutant cells lacking glucose-6-phosphate dehydrogenase are unable to catabolize glucose under these conditions. The mutant cells utilize glucose as effectively as do wild-type cells in the presence of oxygen; under these conditions, glucose is utilized via direct oxidation to gluconate, which is converted to 6-phosphogluconate. The membrane-associated glucose dehydrogenase activity was not formed during anaerobic growth with glucose. Gluconate, the product of the enzyme, appeared to be the inducer of the gluconate transport system, gluconokinase, and membrane-associated gluconate dehydrogenase. 6-Phosphogluconate is probably the physiological inducer of glucokinase, glucose-6-phosphate dehydrogenase, and the dehydratase and aldolase of the Entner-Doudoroff pathway. Nitrate-linked respiration is required for the anaerobic uptake of glucose and gluconate by independently regulated transport systems in cells grown under denitrifying conditions.     

Cloning and characterization of CalS7 from Micromonospora echinospora sp. calichensis as a glucose-1-phosphate nucleotidyltransferase

The deoxysugar biosynthetic gene cluster of calicheamicin contains the calS7, which encodes glucose-1-phosphate nucleotidyltransferase and converts glucose-1-phosphate and nucleotides (NTP) to NDP-glucose and pyrophosphate. calS7 was expressed in Escherichia coli BL21(DE3), and the purified protein had significant thymidylyltransferase and uridylyltransferase activities as well, with some guanidylyltransferase activity but negligible cytidyl and adenyltransferase activity. The functions of thymidylyltransferase and uridylyltransferase were also verified using one-pot enzymatic synthesis of TMK and ACK. The products were analyzed by HPLC and ESI/MS, which showed peaks at m/z = 563 and 565 for TDP-d-glucose and UDP-d-glucose, respectively, in negative mode.     

Enzymes of glucose catabolism in Monilinia fructicola

Summary All enzymes of the Embden-Meyerhof-Parnas pathway were detected in cell-free extracts ofMonilinia fructicola. Hexokinase activity was dependent on the presence of the fluoride ion. The glyceraldehyde-3-phosphate dehydrogenase reaction lasted only a short time. Extracts contained active glucose-6-phosphate and 6-phosphogluconate dehydrogenases of the hexose-monophosphate shunt. No pyruvic dehydrogenase activity could be detected.     

A study of the in-vitro regulation of phosphoenolpyruvate carboxylase from the epidermis of Commelina communis by malate and glucose-6-phosphate

Phosphoenolpyruvate (PEP) carboxylase activity in epidermal extracts of Commelina communis has been compared in the presence of malate and glucose-6-phosphate. The activity of PEP carboxylase was inhibited by increasing malate concentrations at several substrate (PEP) concentrations and changes in both the apparent K _m (PEP) and V _max values in the presence of malate suggested the occurence of mixed-type inhibiton. In the presence of glucose-6-phosphate no increase in enzyme activity was observed, although there was a slight decrease in the K _m (PEP). However, glucose-6-phosphate appeared to alleviate the inhibition caused by malate. The possible implications of these properties in the control of malate production in guard cells is discussed.Abbreviations PEP phosphoenolpyruvate - Glc6P glucose-6-phosphate     

Glucose metabolism by trout (Salmo trutta) red blood cells

Summary Glucose metabolism has been studied in Salmo trutta red blood cells. From non-metabolizable analogue (3-O-methyl glucose and l-glucose) uptake experiments it is concluded that there is no counterpart to the membrane transport system for glucose found in mammalian red blood cells. Once within the cells, glucose is directed to CO₂ and lactate formation through both the Embden-Meyerhoff and hexose monophosphate shunts; lactate appears as the most important endproduct of glucose metabolism in these cells. From experiments under anaerobic conditions, and in the presence of an inhibitor of pyruvate transfer to mitochondria, most of the CO₂ formed appears to derive from the hexose monophosphate pathway. Appreciable O₂ consumption has been detected, but there is no clear relationship between this and substrate metabolism. Key enzymes of glucose metabolism hexokinase, fructose-6-phosphate kinase and, probably, pyruvate kinase are out of equilibrium, confirming their regulatory activity in Salmo trutta red blood cells. The presence of isoproterenol, a catecholamine analogue, induces important changes in glucose metabolism under both aerobic and anaerobic conditions, and increases the production of both CO₂ and lactate. From the data presented, glucose appears to be the major fuel for Salmo trutta red blood cells, showing a slightly different pattern of glucose metabolism from rainbow trout red blood cells.Abbreviations EM Embden-Meyerhoff pathway - G6D glucose-6-phosphate dehydrogenase - GOT glutamate oxalacetate transaminase - GPI glucose phosphate isomerase - HK hexokinase - HMS hexose monophosphate shunt - IP isoproterenol - LDH lactate dehydrogenase - MCB modified Cortland buffer - OMG 3-O-methyl glucose - PFK fructose-6-phosphate kinase - PK pyruvate kinase - RBC red blood cells - TAC tricarboxylic acid cycle     

Construction of a new catabolic pathway for d-fructose in Escherichia coli K12 using an l-sorbose-specific enzyme from Klebsiella pneumoniae

Starting with a fruK (formerly fpk) mutant of Escherichia coli K12 lacking d-fructose-1-phosphate kinase (E.C. 2.7.1.3.), fructose positive derivatives were isolated after introduction of the cloned gene sorE from Klebsiella pneumoniae coding for an l-sorbose-1-phosphate reductase. The new pathway was shwon to proceed from d-fructose via d-fructose-1-phosphate and d-mannitol-1-phosphate to d-fructose 6-phosphate. It involves a transport system and enzymes encoded in the fru and the mtl operons from E. coli K12 as well as in the sor operon from K. pneumoniae respectively.