Metabolism of D-mannose in Aerobacter aerogenes: evidence for a cyclic pathway

Kamel, M. Y. (Michigan State University, East Lansing), and R. L. Anderson. Metabolism of d-mannose in Aerobacter aerogenes: evidence for a cyclic pathway. J. Bacteriol. 92:1689-1697. 1966.-Evidence is presented which suggests a cyclic pathway for the constitutive utilization of d-mannose in extracts of Aerobacter aerogenes PRL-R3. d-Mannose is phosphorylated with d-glucose-6-phosphate to yield d-mannose-6-phosphate and d-glucose. d-Glucose-6-phosphate may be regenerated by isomerization of d-mannose-6-phosphate through d-fructose-6-phosphate, or by phosphorylation of d-glucose with adenosine-5'-triphosphate. The pathway involves the participation of four constitutive enzymes: d-glucose-6-phosphate isomerase, d-mannose-6-phosphate isomerase, a stereospecific d-glucokinase, and a phosphotransferase which phosphorylates d-mannose with d-glucose-6-phosphate, acetyl phosphate, or carbamyl phosphate. The absence of d-mannokinase (adenosine-5'-triphosphate:d-mannose phosphotransferase) activity in extracts of this organism suggests that the pathway may be of functional significance. Also, the pathway accounts for an apparent 2-epimerization of d-mannose to d-glucose that was observed in extracts.     

Substrate specificity of sugar transport by rabbit SGLT1: single-molecule atomic force microscopy versus transport studies

In the apical membrane of epithelial cells from the small intestine and the kidney, the high-affinity Na+/d-glucose cotransporter SGLT1 plays a crucial role in selective sugar absorption and reabsorption. How sugars are selected at the molecular level is, however, poorly understood. Here atomic force microscopy (AFM) was employed to investigate the substrate specificity of rbSGLT1 on the single-molecule level, while competitive-uptake assays with isotope-labeled sugars were performed in the study of the stereospecificity of the overall transport. rbSGLT1-transfected Chinese hamster ovary (CHO) cells were used for both approaches. Evidence of binding of d-glucose to the extracellular surface of rbSGLT1 could be obtained using AFM tips carrying 1-thio-d-glucose coupled at the C1 position to a PEG linker via a vinylsulfon group. Competition experiments with monosaccharides in solution revealed the following selectivity ranking of binding: 2-deoxy-d-glucose >or= 6-deoxy-d-glucose > d-glucose > d-galactose >or= alpha-methyl glucoside; 3-deoxy-d-glucose, d-xylose, and l-glucose did not measurably affect binding. These results were different from those of competitive alpha-methyl glucoside transport assays, where the ranking of inhibition was as follows: d-glucose > d-galactose > 6-deoxy-d-glucose; no uptake inhibition by d-xylose, 3-deoxy-d-glucose, 2-deoxy-d-glucose, or l-glucose was observed. Taken together, these results suggest that the substrate specificity of SGLT1 is determined by different recognition sites: one possibly located at the surface of the transporter and others located close to or within the translocation pathway.     

Specific d-glucose transport in sarcolemma vesicles

The sarcolemmal fraction prepared from rat skeletal muscle consists of osmotically active vesicles that accumulate d-glucose in preference to l-glucose, apparently by facilitated diffusion into intravesicular space. Stereospecific d-glucose uptake by these vesicles is a saturable process, inhibited by phloridzin, by cytochalasin B, and by certain sugars, and enhanced by counterflow. An additional leak pathway permits entry of both d- and l-glucose into the vesicles.Stereospecific d-glucose transport by sarcolemmal vesicles is enhanced to a small extent by insulin, provided the hormone is administered prior to cell disruption. In membranes prepared from insulin-pretreated muscle, Ca²⁺ produces a small further enhancement. Local anesthetics preferentially inhibit stereospecific d-glucose transport. Apparent uptake of both d- and l-glucose is greater when vesicles are suspended in salt solutions rather than sucrose, an effect attributed to increased functional vesicular volume.     

Chemotaxis toward sugars in Escherichia coli

Using a quantitative assay for measuring chemotaxis, we tested a variety of sugars and sugar derivatives for their ability to attract Escherichia coli bacteria. The most effective attractants, i.e., those that have thresholds near 10(-5) M or below, are N-acetyl-d-glucosamine, 6-deoxy-d-glucose, d-fructose, d-fucose, 1-d-glycerol-beta-d-galactoside, galactitol, d-galactose, d-glucosamine, d-glucose, alpha-d-glucose-1-phosphate, lactose, maltose, d-mannitol, d-mannose, methyl-beta-d-galactoside, methyl-beta-d-glucoside, d-ribose, d-sorbitol, and trehalose. Lactose, and probably d-glucose-1-phosphate, are attractive only after conversion to the free monosaccharide, while the other attractants do not require breakdown for taxis. Nine different chemoreceptors are involved in detecting these various attractants. They are called the N-acetyl-glucosamine, fructose, galactose, glucose, maltose, mannitol, ribose, sorbitol, and trehalose chemoreceptors; the specificity of each was studied. The chemoreceptors, with the exception of the one for d-glucose, are inducible. The galactose-binding protein serves as the recognition component of the galactose chemoreceptor. E. coli also has osmotically shockable binding activities for maltose and d-ribose, and these appear to serve as the recognition components for the corresponding chemoreceptors.     

Enzymic methods for the micro assay of D-mannose,D-glucose,D-galactose,and L-fucose from acid hydrolyzates of glycoproteins

Enzymic methods of micro assay have been described for four neutral sugars commonly present in glycoproteins and glycolipids. These assays can be applied to glycoprotein hydrolyzates without prior purification of individual sugars.d-Mannose is assayed by first phosphorylating the sugar in the presence of hexokinase and then measuring the formation of ADP by the use of pyruvate kinase and lactic dehydrogenase. This assay is not specific for d-mannose since both d-glucose and d-glucosamine can be phosphorylated by hexokinase. It is possible to remove d-glucosamine prior to hexokinase treatment by passage through a Dowex 50-X8 (H⁺) column. The effect of d-glucose in the sample can be corrected for by measuring d-glucose with d-glucose-6-phosphate dehydrogenase, an assay which is highly specific for d-glucose.d-Galactose and l-fucose are measured by their respective dehydrogenases. Neither of these dehydrogenases is affected by sugars commonly found in glycoproteins or glycolipids, nor by the presence of a partial acid hydrolyzate of bovine serum albumin. The methods described enable detection of 0.025 μmole of d-mannose, d-glucose, d-galactose, or l-fucose in a glycoprotein digest. The methods can theoretically be made even more sensitive by the use of fluorometric techniques.     

The effect of the strongly bound protein fraction on sugar transport in human erythrocyte ghosts

The protein fraction released from human erythrocyte membranes with 90% acetic acid enhanced the transport of several sugar species when enclosed in erythrocyte ghosts. Both the influx and the efflux of d-glucose were increased so that permeation rather than sugar binding was involved. The permeation increase was selective, being found with d-glucose, d-galactose and d-xylose but not with l-glucose or lactose. The protein-dependent sugar transport was saturable and the incorporation of proteins into the ghost membrane brought J_max to the level corresponding to intact erythrocytes, leaving K_m unchanged.     

Trehalose phosphate synthesis in Streptomyces hygroscopicus: purification of guanosine diphosphate D-glucose: D-glucose-6-phosphate 1-glucosyl-transferase

Guanosine diphosphate d-glucose:d-glucose-6-phosphate 1-glucosyl-transferase was purified approximately 100-fold from extracts of Streptomyces hygroscopicus. The purified enzyme catalyzed the transfer of glucose from guanosine diphosphate-d-glucose to glucose-6-phosphate to form trehalose phosphate and guanosine diphosphate. The enzyme was specific for these two substrates and was stimulated by the addition of magnesium ions. The product was characterized as alpha-alpha-trehalose-6-phosphate by its physical and chemical properties. The enzyme was present in a large number of Streptomyces species, suggesting that this group of organisms synthesized trehalose phosphate in a unique manner. This enzyme was not detected in fungi, since these organisms utilized uridine diphosphate-d-glucose as the glucosyl donor.     

Transport of D-arabinose-5-phosphate and D-sedoheptulose-7-phosphate by the hexose phosphate transport system of Salmonella typhimurium

d-Arabinose-5-phosphate and d-sedoheptulose-7-phosphate were found to be substrates, although not inducers, of the hexose phosphate transport system of Salmonella typhimurium. Transport of these two sugar phosphates by wild-type strains required preinduction of the hexose phosphate transport system. A mutant of S. typhimurium constitutive for this system also transported d-arabinose-5-phosphate and d-sedoheptulose-7-phosphate in a constitutive fashion. Glucose-6-phosphate was a potent competitor of the transport of both d-arabinose-5-phosphate and d-sedoheptulose-7-phosphate. The K(m) values for transport of d-glucose-6-phosphate, d-arabinose-5-phosphate, and d-sedoheptulose-7-phosphate were 0.13, 0.32 and 1.61 mM, respectively. The apparent V(max) values for transport of d-glucose-6-phosphate, d-arabinose-5-phosphate, and d-sedoheptulose-7-phosphate were 6.3, 13.2 and 3.0 nmol per min per 5 x 10(8) bacteria, respectively. d-Ribulose-5-phosphate and d-xylulose-5-phosphate did not inhibit transport of the above substrates, whereas d-ribose-5-phosphate was a weak inhibitor of d-sedoheptulose-7-phosphate transport.     

A facile radiometric technique for measuring ATP

A facile radiometric technique for measuring ATP in samples of biologic origin is presented. The d-glucose-6-phosphate produced by the phosphorylation of excess tritiated d-glucose with crystalline hexokinase is stoichiometrically equivalent to the ATP present. Product is selectively precipitated with ethanolic barium acetate, washed with ethanol and the tritium label counted in a scintillation spectrometer. The technique is especially suitable for the measurement of ATP in large numbers of samples (50–100) and offers acceptable sensitivity down to 62 fmoles.     

Studies on sugar isomerases of Lactobacillus sp.: Whole cell immobilization of d-glucose isomerase

A new soil isolate of Lactobacillus sp. grown in Yamanaka medium under submerged conditions showed the presence of d-glucose, d-xylose and d-ribose isomerases in washed cell suspension and cell free extracts. d-Xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) and d-ribose isomerase (d-ribose ketol-isomerase, EC 5.3.1.20) activities reached a maximum in 48 h of growth and then declined. d-Glucose isomerase (d-glucose 6-phosphate isomerase, d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) activity was maximum after 72 h and remained constant for ～120 h of growth. d-Glucose isomerase activity increased with the increase in number of generations of culture and reached a maximum in 5–6 generations, whereas d-xylose and d-ribose isomerase activities decreased. The washed and starved whole cells could be heat treated and immobilized on the rough surface of glass rods or glass slides using acetone treatment. The heat treated immobilized cells showed only the presence of d-glucose isomerase activity and showed no d-xylose and d-ribose isomerase activities. d-Glucose isomerase activity of heat treated immobilized cells was inhibited less by sorbitol, mannitol, sodium arsenate, cysteine and calcium ions than the free d-glucose isomerase activity in fresh untreated washed whole cells and cell free extracts. EDTA inhibition had the same effect for both forms. Ca²⁺inhibition could be reversed by adding Mg²⁺ions.     

Permeation of macromolecules into polyelectrolyte microcapsules

Polyelectrolyte microcapsules (PEMCs) have been prepared by coating red blood cells with the polyelectrolytes poly(styrenesulfonate), poly(allylamine hydrochloride), and dextran sulfate applying the layer-by-layer technique with subsequent dissolution of the core. The capsule permeability for human serum albumin (HSA) was studied as a function of the ionic strength and pH by means of confocal microscopy. PEMCs produced with dextran sulfate and poly(allylamine hydrochloride) show a significant increase in permeability for HSA at salt concentrations over 1 mM. For PEMCs prepared with poly(styrenesulfonate) and poly(allylamine hydrochloride) the limiting salt concentration is 5 mM. No pH dependence for permeation was observed. A correlation between the permeation and adsorption of HSA on the PEMC walls was investigated. Finally, a mechanism for the permeability, combining electrostatic interactions, and the presence of pores in the polymer layers is presented confirmed by the considerable increase of permeation of charged molecules in the presence of salt and the permeation of neutral molecules regardless of the ionic strength.     

Glucose transport in thymocyte plasma-membrane vesicles

Calf-thymocyte membrane vesicles, prepared by hypotonic lysis and homogenization, were isolated by standard centrifugal techniques designed for enrichment of plasma membrane. At 20°C, these vesicles equilibrated with d-glucose and 3-O-methyl-d-glucose more rapidly than with l-glucose. About 25% of the equilibrium d-sugar space (6 μl/mg protein) was very slowly penetrated by l-glucose ( ). The time course of d-sugar accumulation in excess of l-glucose accumulation indicated that this space equilibrated with d-glucose and 3-O-methyl-d-glucose with half-times of approximately 0.2–0.4 min. The remainder of the equilibrium d-sugar space (about 75%) appeared equally accessible to both glucose isomers ( to 5 min). This was confirmed in studies of efflux from preloaded vesicles, where the d-glucose space fell with a short half-time (0.2 min) to the l-glucose space, after which the two isomers exited with the same half-time. Addition of sucrose to increase osmolarity reduced both spaces (specific and non-specific) in a manner which indicated that little if any of the vesicle sugar was bound. This was confirmed by the fact that equilibrium glucose space was independent of glucose concentration and by the fact that vesicles immediately lost their sugar when diluted with water at 0°C. These data indicate the presence of two vesicle types, discriminant and indiscriminant as regards transport of the glucose isomers. Entry of d-glucose into the discriminant (stereospecific) vesicles was temperature sensitive (Q₁₀ > 2), saturable (K_m 2 mM), and was inhibited by phloretin (K_i < 200 μM), N-ethylmaleimide (K_i < 10 mM) and cytochalasin B (K_i < 2 μM), suggesting that these vesicles contain the plasma-membrane glucose carrier. Entry of l- and d-glucose into the indiscriminant vesicles showed none of these properties. The equilibrium-exchange K_m and V were about five times the entry K_m and V, indicating the substrate loading greatly facilitates carrier translocation, at least in the outward direction.     

The effect of glucose, insulin and noradrenaline on lipolysis and on the concentrations of adenosine 3′:5′-cyclic monophosphate and adenosine 5′-triphosphate in adipose tissue

1. The deoxyfluoro-d-glucopyranose 6-phosphates were prepared from the corresponding deoxyfluoro-d-glucoses and ATP by using hexokinase. 2. 3-Deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-glucose 6-phosphate were substrates for glucose phosphate isomerase, and in addition the products of this reaction, 3-deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-fructose 6-phosphate respectively, were good substrates for phosphofructokinase. 3. Some C-2-substituted derivatives of d-glucose 6-phosphate were found to be competitive inhibitors of glucose phosphate isomerase. 4. The possible role of the hydroxyl groups in the binding of d-glucose 6-phopshate to glucose phosphate isomerase is discussed.     

Transport of d-glucose by brush border membranes isolated from the renal cortex

The transport of d-glucose by brush border membranes isolated from the rabbit renal cortex was studied. At concentrations less than 2 mM, the rate of d-glucose uptake increased linearly with the concentration of the sugar. No evidence was found for a “high-affinity” (μM) saturable site. Saturation was indicated at concentrations of d-glucose greater than 5 mM. The uptake of d-glucose was stereospecific and selectively inhibited by d-galactose and other sugars. Phlorizin inhibited the uptake of d-glucose in the presence and absence of Na⁺. The glycoside was a potent inhibitor of the efflux of d-glucose. Preloading the brush border membrane vesicles with d-glucose, but not with l-glucose, accelerated exchange diffusion of d-glucose. These results demonstrate that the uptake of d-glucose by renal brush borders represents transport into an intravesicular space rather than solely binding. The rate of d-glucose uptake was increased when the Na⁺ in the extravesicular medium was high and the membranes were preloaded with a Na⁺-free medium. The rate of d-glucose uptake was inhibited by preloading the brush border membranes with Na⁺. These results are consistent with the Na⁺ gradient hypothesis for d-glucose transport in the kidney. Thus, the presence of a Na⁺-dependent facilitated transport of d-glucose in isolated renal brush border membranes is indicated. This finding is consistent with what is known of the transport of the sugar in more physiologically intact preparations and suggests that the membranes serve as an effective model system in examining the mechanism of d-glucose transport in the kidney.     

Responsive behavior of tumor-marker-imprinted hydrogels using macromolecular cross-linkers

Tumor-marker-imprinted hydrogels having lectin and antibody molecules as ligands for a tumor-specific marker glycoprotein were strategically prepared by biomolecular imprinting using minute amounts of low-molecular-weight or high-molecular-weight cross-linkers. The tumor-marker-imprinted hydrogels shrank gradually in response to a target glycoprotein, because their apparent cross-linking density increased owing to simultaneous complex formation of lectin and antibody ligands with a target glycoprotein after their ligands dynamically recognized the glycoprotein. The swelling ratio of the tumor-marker-imprinted hydrogel using high-molecular-weight cross-linker with an optimal chain length decreased more drastically than that using a low-molecular-weight cross-linker, but the hydrogel using the cross-linker with a chain that was too long did not exhibit tumor-marker responsive behavior. This paper focuses on the effect of the molecular weight of cross-linkers on the responsive behavior of tumor-marker-imprinted hydrogels having lectin and antibody molecules as ligands. The cross-linker chain length was an important factor in determining the dynamic glycoprotein recognition and responsive behavior of the biomolecule-imprinted hydrogels.     

An abiotic analogue of the nuclear pore complex hydrogel

We describe an abiotic hydrogel that mimics selectivity of the nuclear pore complex. Copolymerization of peptide tetramers (phenylalanine-serine-phenylalanine-glycine, FSFG) with acrylamide results in hydrophobic interactions significant enough to allow the formation of freestanding hydrogel structures. Incorporation of FSFG motifs also renders the hydrogels selective. Selective binding of importins and nuclear transport receptor-cargo complexes is qualitatively demonstrated and compared with polyacrylamide, hydrogels prepared from a control peptide, and hydrogels prepared from the nuclear pore complex protein Nsp1. These abiotic hydrogels will enable further studies of the unique transport mechanisms of the nuclear pore complex and provide an interesting paradigm for the future development of synthetic platforms for separations and selective interfaces.     

Hyperglycemia-induced changes in hepatic membrane fatty acid composition correlate with increased caspase-3 activities and reduced chick embryo viability

Fertile chicken eggs were injected with various concentrations of either d-glucose or l-glucose during the first three days of embryonic development. The exogenous glucose concentrations ranged from 0 to 18.58 micromol/kg egg. At 18 days of development (theoretical stage 44), brains, livers, and blood from chorio-allantoic vessels were isolated from living embryos. Exogenous d-glucose and l-glucose caused increased plasma d-glucose levels, increased plasma alanine aminotransferase (ALT) activities, and decreased embryo viability. Embryo viability was monitored by a reduction in the percentage of living embryos at theoretical stage 44, reduced embryo masses, reduced brain masses, and reduced liver masses. When compared to controls, embryonic exposure to either exogenous d-glucose or l-glucose caused increased caspase-3 activities and increased lipid hydroperoxide (LPO) levels in both brain and liver tissues. Because lipid hydroperoxides are lipid peroxidation intermediates that result in the attack of any unsaturated neutral lipid or unsaturated phospholipid, the effect of exogenous glucose on hepatic membrane fatty acid composition was studied. Exogenous glucose (either d-glucose or l-glucose) promoted reduced levels of several unsaturated, long-chain fatty acids and increased levels of saturated, short-chain fatty acids within hepatic membranes. Exogenous-glucose induced decreases in the ratios of unsaturated/saturated fatty acids and long-chain/short-chain fatty acids within hepatic membranes which strongly correlated with glucose-induced increases in plasma ALT activities and moderately correlated to hepatic LPO levels. These observations are consistent with the hypothesis that embryonic hyperglycemia promotes hepatic membrane lipid peroxidation and hepatic cell death.     

Efficient production of 2-deoxy-scyllo-inosose from d-glucose by metabolically engineered recombinant Escherichia coli

2-Deoxy-scyllo-inosose (DOI) is a six-membered carbocycle formed from d-glucose-6-phosphate catalyzed by 2-deoxy-scyllo-inosose synthase (DOIS), a key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics. DOI is valuable as a starting material for the benzene-free synthesis of catechol and other benzenoids. We constructed a series of metabolically engineered Escherichia coli strains by introducing a DOIS gene (btrC) from Bacillus circulans and disrupting genes for phosphoglucose isomerase, d-glucose-6-phosphate dehydrogenase, and phosphoglucomutase (pgi, zwf and pgm, respectively). It was found that deletion of the pgi gene, pgi and zwf genes, pgi and pgm genes, or all pgi, zwf and pgm genes significantly improved DOI production by recombinant E. coli in 2YTG medium (3% glucose) up to 7.4, 6.1, 11.6, and 8.4 g l(-1), respectively, compared with that achieved by wild-type recombinant E. coli (1.5 g l(-1)). Moreover, E. coli mutants with disrupted pgi, zwf and pgm genes showed strongly enhanced DOI productivity of up to 29.5 g l(-1) (99% yield) in the presence of mannitol as a supplemental carbon source. These results demonstrated that DOI production by metabolically engineered recombinant E. coli may provide a novel, efficient approach to the production of benzenoids from renewable d-glucose.     

Purification of His-tagged proteins using Ni2+-poly(2-acetamidoacrylic acid) hydrogel

In this study, a new matrix for immobilized metal affinity chromatography (IMAC) using poly(2-acetamidoacrylic acid) (PAAA) hydrogels complexed with Ni(2+) was developed for the purification of the recombinant histidine-tagged green fluorescence protein (His6-GFP). The Ni(2+)-complexed PAAA hydrogel was prepared by polymerizing 2-acetamidoacrylic acid (AAA) and 2,2'-[(1,4-dioxo-1,4-butanediyl)diamino] bis(2-propenoic acid) (DBDBPA) with potassium persulfate in DMSO, followed by Ni(2+) complexation. Confocal laser scanning microscopy was used to determine the binding of His6-GFP to the Ni(2+)-PAAA hydrogel in three-dimensional space. Photoluminescence spectroscopy revealed an 81% binding efficiency of His6-GFP to the Ni(2+)-PAAA hydrogel yielded with a recovery of 59%. The specificity of His6-GFP binding to Ni(2+)-PAAA hydrogel was compared with that of the PAAA hydrogel without Ni(2+). His6-GFP was purified directly from the cell lysate with Ni(2+)-PAAA hydrogel matrix but the PAAA hydrogel without Ni(2+) had no effect. The major advantage of the Ni(2+)-PAAA hydrogel system over current methods, such as Ni-nitrilotriacetic acid (NTA) agarose beads, was the simple and low-cost procedure for preparing the matrix.     

Preparation of photolithographically patterned inverse opal hydrogel microstructures and its application to protein patterning

Protein pattern has played an important role in biosensors, bioMEMS, tissue engineering, fundamental studies of cell biology, and basic proteomics research. Here, we developed a straightforward and effective protein patterning technique using macroporous poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel micropatterns as a three-dimensional (3D) template for protein immobilization. Micropatterns of macroporous hydrogels with inverse opal structures were prepared on poly(ethylene glycol) (PEG)-coated silicon substrates by combining a colloidal crystal templating method with photopatterning. The resultant inverse opal hydrogel (IOH) micropatterns were modified with 3-aminopropyltriethoxysilane using the hydroxyl groups in PHEMA for the covalent immobilization of proteins. Proteins were selectively immobilized only on the hydrogel micropatterns, while the PEG regions served as an effective barrier to protein adsorption. Because of their highly ordered and interconnected 3D macroporous structures and large internal surface areas, protein loading in the IOH micropattern was about six times greater than that on a non-porous hydrogel micropattern, which consequently improved the protein activity. The porosity of the hydrogel micropatterns could be controlled using different sizes of colloidal nanoparticles, and using smaller nanoparticles produced hydrogel micropatterns with higher protein loading capacities and activities. To demonstrate the potential use of IOH micropatterns in biosensor systems, biotin was micropatterned on the hydrogels and the specific binding of streptavidin was successfully assayed using IOH micropatterns with better fluorescence signals and sensitivity than that of the corresponding non-porous hydrogel micropatterns.