Structural requirements for active intestinal transport. The nature of the carrier–sugar bonding at C-2 and the ring oxygen of the sugar

Several weakly transported sugars were tested for transport by the Na⁺-dependent sugar carrier with slices of everted hamster intestinal tissue. Sugars were assumed to be transported by this carrier if the accumulation was diminished in the absence of Na⁺ and in the presence of the competitive inhibitor 1,5-anhydro-d-glucitol. The extent of accumulation was correlated with the number of hydroxyl groups in the d-gluco configuration if the ring oxygen was placed in the normal d-glucose position. 5-Thio-d-glucose, with a sulphur atom in the ring, was transported at about the same rate as d-glucose and had a similar K_i for d-galactose transport, but myoinositol was poorly accumulated. It is suggested that there is no hydrogen bonding at the ring oxygen atom, but that the oxygen atom is found at this position as a result of steric constraints. No sugar without a hydroxyl group in the d-gluco position at C-2 of the sugar, including d-mannose, 2-deoxy-d-glucose, 2-chloro-2-deoxy-d-glucose and 2-deoxy-2-fluoro-d-glucose, was transported by the Na⁺-dependent carrier, but these sugars and l-fucose weakly and competitively inhibit the Na⁺-dependent accumulation of l-glucose into slices of everted hamster intestinal tissue. It is concluded that the bond between the carrier and C-2 of the sugar may be covalent, and a possible mechanism for active intestinal transport is proposed.     

Sugar transport across the peritubular face of renal cells of the flounder

The transport of some sugars at the antiluminal face of renal cells was studied using teased tubules of flounder (Pseudopleuronectes americanus). The analytical procedure allowed the determination of both free and total (free plus phosphorylated) tissue sugars. The inulin space of the preparation was 0.333 ± 0.017 kg/kg wet wt (7 animals, 33 analyses). The nonmetabolizable α-methyl-D-glucoside entered the cells by a carrier-mediated (phloridzin-sensitive), ouabain-insensitive process. The steady-state tissue/medium ratio was systematically below that for diffusion equilibrium. D-Glucose was a poor inhibitor of α-methyl-glucoside transport, D-galactose was ineffective. The phloridzin-sensitive transport processes of 2-deoxy-D-glucose,D-galactose,and 2-deoxy-D-galactose were associated with considerable phosphorylation. Kinetic evidence suggested that these sugars were transported in free form and subsequently were phosphorylated. 2-Deoxy-D-glucose accumulated in the cells against a slight concentration gradient. This transport was greatly inhibited by D-glucose, whereas α-methyl-glucoside and also D-galactose and its 2-deoxy-derivative were ineffective. D-Galactose and 2-deoxy-D-galactose mutually competed for transport; D-glucose, 2-deoxy-D-glucose, and α-methyl-D-glucoside were ineffective. Studies using various sugars as inhibitors suggest the presence of three carrier-mediated pathways of sugar transport at the antiluminal cell face of the flounder renal tubule: the pathway of α-methyl-D-glucoside (not shared by D-glucose); the pathway commonly shared by 2-deoxy-D-glucose and D-glucose; the pathway shared by D-galactose and 2-deoxy-D-galactose.     

Transport of glucose and galactose in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25° was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S]_i) and extracellular ([S]_o) glucose concentrations was increased by 0·4mm-phlorrhizin and 0·3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S]_i/[S]_o fell below 1·0 only at [S]_o higher than 0·5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K_m 1·16mm; V_max. 4·5μmoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0·1mm-2,4-dinitrophenol, 0·4mm-phlorrhizin and by the absence of external Na⁺. 6. The kinetic parameters of galactose entry into the cells were: K_m 1·5mm; V_max 10μmoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0°, was inhibited by 0·4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na⁺-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na⁺-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

A Novel Reaction of Sugars with Anion Radical of Carbon Dioxide Produced from Formate

Radiolysis of some monosaccharides (fructose, glucose and ribose) in air-free condition was markedly enhanced by the addition of formate at concentrations above 20 mm, while it was inhibited at concentrations below 20 mm. The following compounds were detected in the irradiated sugar solutions containing excess formate (100mm): 1-Deoxy-d-arabinohexulose (1, G=4.4) and 1,3- dideoxy-d-erythrohexulose (2, G= 1.3) from fructose; 2-deoxy-d-ribose (3, G=2.3) and 2-deoxyribitol (4, G =0.6) from ribose; and 2-deoxy-d-glucose (5, G=0.5) and 2-deoxy-d-glucitol (6, G=0.4) from glucose. A mechanism for radiolytic formation of the products was proposed, based on interaction of - formed from formate with sugars.     

In Vitro Analysis of the H-Hexose Symporter on the Plasma Membrane of Sugarbeets (Beta vulgaris L.)

The mechanism of hexose transport into plasma membrane vesicles isolated from mature sugarbeet leaves (Beta vulgaris L.) was investigated. The initial rate of glucose uptake into the vesicles was stimulated approximately fivefold by imposing a transmembrane pH gradient (ΔpH), alkaline inside, and approximately fourfold by a negative membrane potential (ΔΨ), generated as a K⁺-diffusion potential, negative inside. The -fold stimulation was directly related to the relative ΔpH or ΔΨ gradient imposed, which were determined by the uptake of acetate or tetraphenylphosphonium, respectively. ΔΨ- and ΔpH-dependent glucose uptake showed saturation kinetics with a K_m of 286 micromolar for glucose. Other hexose molecules (e.g. 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-mannose) were also accumulated into plasma membrane vesicles in a ΔpH-dependent manner. Inhibition constants of a number of compounds for glucose uptake were determined. Effective inhibitors of glucose uptake included: 3-O-methyl-d-glucose, 5-thio-d-glucose, d-fructose, d-galactose, and d-mannose, but not 1-O-methyl-d-glucose, d- and l-xylose, l-glucose, d-ribose, and l-sorbose. Under all conditions of proton motive force magnitude and glucose and sucrose concentration tested, there was no effect of sucrose on glucose uptake. Thus, hexose transport on the sugarbeet leaf plasma membrane was by a H⁺-hexose symporter, and the carrier and possibly the energy source were not shared by the plasma membrane H⁺-sucrose symporter.     

Structural Features of Sugars That Trigger or Support Conidial Germination in the Filamentous Fungus Aspergillus niger

The asexual spores (conidia) of Aspergillus niger germinate to produce hyphae under appropriate conditions. Germination is initiated by conidial swelling and mobilization of internal carbon and energy stores, followed by polarization and emergence of a hyphal germ tube. The effects of different pyranose sugars, all analogues of d-glucose, on the germination of A. niger conidia were explored, and we define germination as the transition from a dormant conidium into a germling. Within germination, we distinguish two distinct stages, the initial swelling of the conidium and subsequent polarized growth. The stage of conidial swelling requires a germination trigger, which we define as a compound that is sensed by the conidium and which leads to catabolism of d-trehalose and isotropic growth. Sugars that triggered germination and outgrowth included d-glucose, d-mannose, and d-xylose. Sugars that triggered germination but did not support subsequent outgrowth included d-tagatose, d-lyxose, and 2-deoxy-d-glucose. Nontriggering sugars included d-galactose, l-glucose, and d-arabinose. Certain nontriggering sugars, including d-galactose, supported outgrowth if added in the presence of a complementary triggering sugar. This division of functions indicates that sugars are involved in two separate events in germination, triggering and subsequent outgrowth, and the structural features of sugars that support each, both, or none of these events are discussed. We also present data on the uptake of sugars during the germination process and discuss possible mechanisms of triggering in the absence of apparent sugar uptake during the initial swelling of conidia.     

Sugar uptake and starch biosynthesis by slices of developing maize endosperm

¹⁴C-Sugar uptake and incorporation into starch by slices of developing maize (Zea mays L.) endosperm were examined and compared with sugar uptake by maize endosperm-derived suspension cultures. Rates of sucrose, fructose, and d- and l-glucose uptake by slices were similar, whereas uptake rates for these sugars differed greatly in suspension cultures. Concentration dependence of sucrose, fructose, and d-glucose uptake was biphasic (consisting of linear plus saturable components) with suspension cultures but linear with slices. These and other differences suggest that endosperm slices are freely permeable to sugars. After diffusion into the slices, sugars were metabolized and incorporated into starch. Starch synthesis, but not sugar accumulation, was greatly reduced by 2.5 millimolar p-chloromercuribenzenesulfonic acid and 0.1 millimolar carbonyl cyanide m-chlorophenylhydrazone. Starch synthesis was dependent on kernel age and incubation temperature, but not on external pH (5 through 8). Competing sugars generally did not affect the distribution of ¹⁴C among the soluble sugars extracted from endosperm slices incubated in ¹⁴C-sugars. Competing hexoses reduced the incorporation of ¹⁴C into starch, but competing sucrose did not, suggesting that sucrose is not a necessary intermediate in starch biosynthesis. The bidirectional permeability of endosperm slices to sugars makes the characterization of sugar transport into endosperm slices impossible, however the model system is useful for experiments dealing with starch biosynthesis which occurs in the metabolically active tissue.     

Substrate Specificity of the H-Sucrose Symporter on the Plasma Membrane of Sugar Beets (Beta vulgaris L.) : Transport of Phenylglucopyranosides

Previous results (TJ Buckhout, Planta [1989] 178: 393-399) indicated that the structural specificity of the H⁺-sucrose symporter on the plasma membrane from sugar beet leaves (Beta vulgaris L.) was specific for the sucrose molecule. To better understand the structural features of the sucrose molecule involved in its recognition by the symport carrier, the inhibitory activity of a variety of phenylhexopyranosides on sucrose uptake was tested. Three competitive inhibitors of sucrose uptake were found, phenyl-α-d-glucopyranoside, phenyl-α-d-thioglucopyranoside, and phenyl-α-d-4-deoxythioglucopyranoside (PDTGP; K_i = 67, 180, and 327 micromolar, respectively). The K_m for sucrose uptake was approximately 500 micromolar. Like sucrose, phenyl-α-d-thioglucopyranoside and to a lesser extent, PDTGP induced alkalization of the external medium, which indicated that these derivatives bound to and were transported by the sucrose symporter. Phenyl-α-d-3-deoxy-3-fluorothioglucopyranoside, phenyl-α-d-4-deoxy-4-fluorothioglucopyranoside, and phenyl-α-d-thioallopyranoside only weakly but competively inhibited sucrose uptake with K_i values ranging from 600 to 800 micromolar, and phenyl-α-d-thiomannopyranoside, phenyl-β-d-glucopyranoside, and phenylethyl-β-d-thiogalactopyranoside did not inhibit sucrose uptake. Thus, the hydroxyl groups of the fructose portion of sucrose were not involved in a specific interaction with the carrier protein because phenyl and thiophenyl derivatives of glucose inhibited sucrose uptake and, in the case of phenyl-α-d-thioglucopyranoside and PDTGP, were transported.     

Characterization of the hexose transport system in maize root tips

Sugar-depleted excised maize (Zea mays L.) root tips were used to study the kinetics and the specificity of hexose uptake. It was found that difficulties induced by bulk diffusion and penetration barriers did not exist with root tips. Several lines of evidence indicate the existence of a complex set of uptake systems for hexoses showing an overall biphasic dependence on external sugar concentrations. The results suggest that the high and the low affinity components might be located on the same carrier. One uptake system was specific for fructose, but the high affinity component was repressed by high concentrations of external glucose. A second system was specific for glucose and its analogs (2-deoxy-d-glucose and 3-O-methyl-d-glucose), and a third one, more complex, had a high affinity for glucose and its analogs but could transport fructose when glucose was not present in the external solution. A simple method is proposed to determine the inhibitor constants in competition experiments.     

Membrane transport of sugars in cell suspensions of sugarcane: I. Evidence for sites and specificity

Sugar uptake by sugarcane cells in suspension culture was measured over short incubation time spans (5 seconds to 4 minutes), and membrane transport rates were calculated. A relatively high proportion of labeled products in cell extracts after incubation of cells with ¹⁴C-glucose for 5 seconds was sugar phosphates (56%); fructose and sucrose began to appear after 15 and 30 seconds, respectively. Galactose and 3-O-methylglucose competed appreciably with glucose uptake, but ketohexoses and pentoses did not; there was no detectable uptake of sucrose. It is postulated that besides endogenous phosphorylation and further metabolism of glucose the configuration of the hydroxyl on the carbon-2 may be important for efficient membrane transport. The cells had a particularly high affinity for glucose and 3-O-methylglucose (Km = 15 and 16 μm, respectively).     

Enzymes of sucrose breakdown in soybean nodules: alkaline invertase

The specific activities of acid and alkaline invertases (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26), sucrose synthase (UDPglucose: d-fructose 2-α-d-glucosyltransferase, EC 2.4.1.13), hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1), and fructokinase (ATP: d-fructose 6-phosphotransferase, EC 2.7.1.4) were determined in soybean (Glycine max L. Merr cv Williams) nodules at different stages of development and, for comparison, in roots of nonnodulated soybeans. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodules, but there was only a small amount of acid invertase present. The nodules contained more phosphorylating activity with fructose than glucose. Essentially all of the alkaline invertase, sucrose synthase, and fructokinase were in the soluble fraction of nodule extracts whereas hexokinase was in the bacteroid, plant particulate, and soluble fractions.     

Hyperinduction of pectate lyase in Dickeya chrysanthemi EC16 by plant-derived sugars

Pectate lyase (Pel) synthesis in Dickeya chrysanthemi has been reported to be hyperinduced in planta and also in the medium containing plant extract in addition to polygalacturonate. In this study, the major components of Pel-hyperinducing fractions were found to be glucose, fructose, and sucrose by TLC and NMR. From the analysis of the sugars and their derivatives, it was found that acyclic d-hexoses with the trans relationship between C-3 and C-5 hydroxyl groups were found to be basic structure required for hyperinducing the expression of a major isozyme in infected plants (i.e. pelE). From the fact that some non-metabolizable sugars such as 2-deoxy-d-glucose and d-fucose could lead to hyperinduction and that the hyperinduction was observed only in the medium containing low concentration (<0.25%) but not higher of the sugars was added, these sugars may be considered to participate in hyperinduction as the signal rather than through their metabolism.     

Uptake of monosaccharides by guinea-pig cerebral-cortex slices

By the use of 1mm-iodoacetate to inhibit glycolysis in guinea-pig cerebral tissue slices, the kinetics of the uptake of monosaccharides on transfer of tissue from 0° to 37° were studied. d-Ribose, d-galactose, d-mannose, l-sorbose, and d-fructose showed diffusion kinetics, whereas 2-deoxy-d-glucose, d-glucose, d-arabinose and d-xylose showed saturation kinetics.     

Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives

A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-β-d-glucoside, resveratrol 4′-O-β-d-glucoside, resveratrol 3,5-O-β-d-diglucoside, and resveratrol 3,5,4′-O-β-d-triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3-O-β-d-2-deoxyglucoside and resveratrol 3,5-O-β-d-di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4′-O-β-d-galactoside, resveratrol 4′-O-β-d-viosaminoside, resveratrol 3-O-β-l-rhamnoside, and resveratrol 3-O-β-l-fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.     

Inhibitory Effect of Carbohydrate on Flowering in Lemna perpusilla: II. Reversal by Glycine and l-Aspartate. Correlation with Reduced Levels of beta-Carotene and Chlorophyll

Flowering of Lemna perpusilla strain 6746 grown on 0.1 strength Hutner's medium in short days was inhibited by sucrose, glucose, fructose, and mannose, but not by various other sugars or metabolic intermediates. Only those sugars that inhibited flowering supported heterotrophic growth. Experiments with a single inductive long night indicated that an early stage in flowering was the sugar-sensitive process. Inhibition of flowering by carbohydrates was accompanied by reduced levels of chlorophyll and β-carotene. The inhibitory effects of carbohydrates on flowering were partially reversed by iminodiacetate, glycine, and l-aspartate but not by d-aspartate, ethylenediaminetetraacetate, acetate, δ-aminolevulinic acid, or mevalonic acid. The possibility is discussed that carbohydrate repression of flowering and of chloroplast pigments resulted from inadequate levels of amino acids.     

The incorporation of labelled amino sugars by Bacillus subtilis

1. Glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] of Bacillus subtilis has been partially purified. Its K_m is 3·0mm. 2. Extracts of B. subtilis contain N-acetylglucosamine 6-phosphate deacetylase (K_m 1·4mm), glucosamine 1-phosphate acetylase and amino sugar kinases (EC 2.7.1.8 and 2.7.1.9). 3. Glucosamine 6-phosphate synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) is repressed by growth of B. subtilis in the presence of glucosamine, N-acetylglucosamine, N-propionylglucosamine or N-formylglucosamine. Glucosamine 6-phosphate deaminase and N-acetylglucosamine 6-phosphate deacetylase are induced by N-acetylglucosamine. Amino sugar kinases are induced by glucose, glucosamine and N-acetylglucosamine. The synthesis of glucosamine 1-phosphate acetylase is unaffected by amino sugars. 4. Glucose in the growth medium prevents the induction of glucosamine 6-phosphate deaminase and of N-acetylglucosamine 6-phosphate deacetylase caused by N-acetylglucosamine; glucose also alleviates the repression of glucosamine 6-phosphate synthetase caused by amino sugars. 5. Glucosamine 6-phosphate deaminase increases in bacteria incubated beyond the exponential phase of growth. This increase is prevented by glucose.     

Further studies on the regulation of amino sugar metabolism in Bacillus subtilis

1. Glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] of Bacillus subtilis has been partially purified. Its K_m is 3·0mm. 2. Extracts of B. subtilis contain N-acetylglucosamine 6-phosphate deacetylase (K_m 1·4mm), glucosamine 1-phosphate acetylase and amino sugar kinases (EC 2.7.1.8 and 2.7.1.9). 3. Glucosamine 6-phosphate synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) is repressed by growth of B. subtilis in the presence of glucosamine, N-acetylglucosamine, N-propionylglucosamine or N-formylglucosamine. Glucosamine 6-phosphate deaminase and N-acetylglucosamine 6-phosphate deacetylase are induced by N-acetylglucosamine. Amino sugar kinases are induced by glucose, glucosamine and N-acetylglucosamine. The synthesis of glucosamine 1-phosphate acetylase is unaffected by amino sugars. 4. Glucose in the growth medium prevents the induction of glucosamine 6-phosphate deaminase and of N-acetylglucosamine 6-phosphate deacetylase caused by N-acetylglucosamine; glucose also alleviates the repression of glucosamine 6-phosphate synthetase caused by amino sugars. 5. Glucosamine 6-phosphate deaminase increases in bacteria incubated beyond the exponential phase of growth. This increase is prevented by glucose.     

Characteristics of a Galactose-adapted Sugarcane Cell Line Grown in Suspension Culture

Although d-galactose is normally toxic to sugarcane (Saccharum sp.) cells, a cell line that grows on 100 mm galactose has been propagated. Nonadapted cells in a medium containing galactose instead of sucrose accumulate UDP-galactose; these cells also have much lower UDP-galactose 4-epimerase (EC 5.1.3.2) activity than do adapted cells. This enzyme may determine whether or not galactose will cause toxicity symptoms to develop. The growth rate of galactose-adapted cells is similar to most cell lines on several other carbohydrates. The galactose-adapted cells are also similar to sucrose stock cells in cell wall composition and sugar phosphate concentrations, but, like the nonadapted cells, accumulate free galactose.     

Purification and Characterization of a Trehalose Synthase from the Basidiomycete Grifola frondosa

A trehalose synthase (TSase) that catalyzes the synthesis of trehalose from d-glucose and α-d-glucose 1-phosphate (α-d-glucose 1-P) was detected in a basidiomycete, Grifola frondosa. TSase was purified 106-fold to homogeneity with 36% recovery by ammonium sulfate precipitation and several steps of column chromatography. The native enzyme appears to be a dimer since it has apparent molecular masses of 120 kDa, as determined by gel filtration column chromatography, and 60 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although TSase catalyzed the phosphorolysis of trehalose to d-glucose and α-d-glucose 1-P, in addition to the synthesis of trehalose from the two substrates, the TSase equilibrium strongly favors trehalose synthesis. The optimum temperatures for phosphorolysis and synthesis of trehalose were 32.5 to 35°C and 35 to 37.5°C, respectively. The optimum pHs for these reactions were 6.5 and 6.5 to 6.8, respectively. The substrate specificity of TSase was very strict: among eight disaccharides examined, only trehalose was phosphorolyzed, and only α-d-glucose 1-P served as a donor substrate with d-glucose as the acceptor in trehalose synthesis. Two efficient enzymatic systems for the synthesis of trehalose from sucrose were identified. In system I, the α-d-glucose 1-P liberated by 1.05 U of sucrose phosphorylase was linked with d-glucose by 1.05 U of TSase, generating trehalose at the initial synthesis rate of 18 mmol/h in a final yield of 90 mol% under optimum conditions (300 mM each sucrose and glucose, 20 mM inorganic phosphate, 37.5°C, and pH 6.5). In system II, we added 1.05 U of glucose isomerase and 20 mM MgSO₄ to the reaction mixture of system I to convert fructose, a by-product of the sucrose phosphorylase reaction, into glucose. This system generated trehalose at the synthesis rate of 4.5 mmol/h in the same final yield.Trehalose (1-α-d-glucopyranosyl-α-d-glucopyranoside) is a nonreducing disaccharide with an α,α-1,1 glycosidic linkage and is widely distributed in plants, insects, fungi, yeast, and bacteria (7). Due to the absence of reducing ends in trehalose, it is highly resistant to heat, pH, and Maillard’s reaction (24). In trehalose-producing organisms, this compound may serve as an energy reserve, a buffer against stresses such as desiccation and freezing, and a protein stabilizer (5, 7, 26, 31, 32). If trehalose can be produced economically, then it has potential commercial applications as a sweetener, a food stabilizer, and an additive in cosmetics and pharmaceuticals (6, 25). Recently, trehalose production through fermentation of yeast (17) and Corynebacterium (30), enzymatic processes from starch (18, 34) and maltose (19, 22, 23, 33), and extraction from transformed plants (10) has been reported.Our approach to trehalose production is to use an enzymatic process to produce trehalose from sucrose, one of the least expensive sugars. Since sucrose is efficiently converted to α-d-glucose 1-phosphate (α-d-glucose 1-P) and fructose by sucrose phosphorylase (SPase), we screened microorganisms for an enzyme that converts α-d-glucose 1-P to trehalose on the assumption that the combination of the putative trehalose synthase (TSase) and SPase would convert sucrose into trehalose. Although similar enzyme activities have been reported in the basidiomycete Flammulina velutipes (11) and in the yeast Pichia fermentans (27), these enzymes have not been well characterized.Our objectives were (i) to screen microorganisms, primarily fungi, for TSase activity; (ii) to purify and characterize the TSase; (iii) to identify the enzymatic process by which trehalose is produced from sucrose; and (iv) to identify an enzymatic process for production of trehalose from sucrose in which the fructose component is also converted to trehalose.     

The use of potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Spectrophotometric measurements

Renal transport of four different categories of organic solutes, namely sugars, neutral amino acids, monocarboxylic acids and dicarboxylic acids, was studied by using the potential-sensitive dye 3,3′-diethyloxadicarbocyanine iodide in purified luminal-membrane and basolateral-membrane vesicles isolated from rabbit kidney cortex. Valinomycin-induced K⁺ diffusion potentials resulted in concomitant changes in dye–membrane-vesicle absorption spectra. Linear relationships were obtained between these changes and depolarization and hyperpolarization of the vesicles. Addition of d-glucose, l-phenylalanine, succinate or l-lactate to luminal-membrane vesicles, in the presence of an extravesicular>intravesicular Na⁺ gradient, resulted in rapid transient depolarization. With basolateral-membrane vesicles no electrogenic transport of d-glucose or l-phenylalanine was observed. Spectrophotometric competition studies revealed that d-galactose is electrogenically taken up by the same transport system as that for d-glucose, whereas l-phenylalanine, succinate and l-lactate are transported by different systems in luminal-membrane vesicles. The absorbance changes associated with simultaneous addition of d-glucose and l-phenylalanine were additive. The uptake of these solutes was influenced by the presence of Na⁺-salt anions of different permeabilities in the order: Cl⁻>SO₄²⁻>gluconate. Addition of valinomycin to K⁺-loaded vesicles enhanced uptake of d-glucose and l-phenylalanine in the presence of an extravesicular>intravesicular Na⁺ gradient. Gramicidin or valinomycin plus nigericin diminished/abolished electrogenic solute uptake by Na⁺- or Na⁺+K⁺-loaded vesicles respectively. These results strongly support the presence of Na⁺-dependent renal electrogenic transport of d-glucose, l-phenylalanine, succinate and l-lactate in luminal-membrane vesicles.