Labeling pattern of translocated stachyose in squash

One mature blade of each squash plant was continuously labeled with ¹⁴CO₂ for 15, 30, or 70 minutes in light. The ethanol soluble materials from serial sections of petioles were extracted and separated by paper chromatography. The ratios of label in the various components of this fraction were determined. Stachyose, which contained the major portion of the label of this fraction, was hydrolyzed and the resultant hydrolysate was separated by paper chromatography. Specific activities of the hexoses derived from stachyose were determined. It was found that the glucose and fructose moieties of stachyose became labeled at the same rates; however, the galactose moiety became labeled more rapidly.     

Transport of Stachyose and Sucrose by Vacuoles of Japanese Artichoke (Stachys sieboldii) Tubers

Vacuoles are the stores for large amounts of stachyose [αgal (1,6) αgal (1,6) αglc (1,2) βfru] in tubers of Japanese artichoke (Stachys sieboldii). The uptake of stachyose by these vacuoles was examined and compared with that of sucrose. The uptake mechanisms of both sugars were quite similar. The kinetics showed a single saturable response to increasing external concentrations of ¹⁴C-sugars with similar apparent K_m values of about 50 and 30 millimolar for stachyose and sucrose, respectively. The uptake rates, however, were always higher for stachyose than for sucrose. Stachyose and sucrose uptake was inhibited by fructose and raffinose, and, reciprocally, by sucrose and stachyose, but not by glucose or galactose. The main structural feature common to all sugars recognized by the uptake systems seems to be a terminal fructosyl residue. The uptake of both sugars was stimulated by Mg-ATP and inorganic pyrophosphate, suggesting a proton-sugar antiport system. The possibility that stachyose and sucrose might be transported by the same carrier is discussed.     

Changes in concentrations of soluble carbohydrates during germination of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. seeds in relation to ethylene,gibberellin A<Subscript>3</Subscript> and methyl jasmonate

Unimbibed Amaranthus caudatus seeds were found to contain stachyose, raffinose, verbascose, sucrose, galactinol, myo-inositol, glucose and fructose, while no galactose, maltose and maltotriose was detected. During imbibition, seed concentrations of verbascose, stachyose, raffinose, galactinol, myo-inositol (temporary) and fructose (transient) were observed to decrease; concentrations of galactose and maltose remained fairly constant, while those of sucrose, glucose and maltotriose increased, the increase in sucrose concentration was only temporary. Effects of gibberellin A₃ (GA₃) at 3 × 10⁻⁴ M and ethephon at 3 × 10⁻⁴ M alone or in the presence of methyl jasmonate (Me-JA) at 10⁻³ M on concentrations of soluble sugars during germination of A. caudatus seeds were examined. Me-JA was found to inhibit seed germination and fresh weight of the seeds, but did not affect sucrose, myo-inositol, galactose and maltose concentrations during imbibition for up to 20 h. The exogenously applied GA₃ was observed to enhance germination, stachyose breakdown and glucose concentration after 20 h of incubation. Ethephon stimulated seed germination as well as utilisation of stachyose, galactinol (both after 14 and 20 h) and raffinose (after 14 h of incubation). Although the stimulatory effect of either GA₃ or ethephon on seed germination was blocked by Me-JA; these stimulators increased mobilisation of raffinose and stachyose, but only ethephon enhanced both glucose and fructose after 14 and/or 20 h of incubation in the presence of Me-JA. The maltose concentration was increased by both GA₃ and ethephon alone and in the presence of Me-JA. Of the growth regulators studied, ethephon alone and/or in combination with Me-JA significantly increased the concentrations of glucose, fructose, galactose, maltose and maltotriose. The differences in sugar metabolism appear to be linked to ethylene or GA₃ applied simultaneously with Me-JA.     

Sugar synthesis and phloem loading in Coleus blumei leaves

Sugar-synthesis and -transport patterns were analyzed in Coleus blumei Benth. leaves to determine where galactinol, raffinose, and stachyose are made and whether phloem loading includes an apoplastic (extracellular) step or occurs entirely within the symplast (plasmodesmata-connected cytoplasm). To clarify the sequence of steps leading to stachyose synthesis, a pulse (15 s) of ¹⁴CO₂ was given to attached leaves followed by a 5-s to 20-min chase: sucrose was rapidly labeled while galactinol, raffinose and stachyose were labeled more slowly and, within the first few minutes, to approximately the same degree. Leaf tissue was exposed to either ¹⁴CO₂ or [¹⁴C]glucose to identify the sites of synthesis of the different sugars. A 2-min exposure of peeled leaf tissue to [¹⁴C]glucose resulted in preferential labeling of the minor veins, as opposed to the mesophyll; galactinol, raffinose and stachyose were more heavily labeled than sucrose in these preparations. In contrast, when leaf tissue was exposed to ¹⁴CO₂ for 2 min for preferential labeling of the mesophyll, sucrose was more heavily labeled than galactinol, raffinose or stachyose. We conclude that sucrose is synthesized in mesophyll cells while galactinol, raffinose and stachyose are made in the minorvein phloem. Competition experiments were performed to test the possibility that phloem loading involves monosaccharide uptake from the apoplast. Two saturable monosaccharide carriers were identified, one for glucose, galactose and 3-O-methyl glucose, and the other for fructose. Washing the apoplast of peeled leaf pieces with buffer or saturating levels of 3-O-methyl glucose, after providing a pulse of ¹⁴CO₂, did not inhibit vein loading or change the composition of labeled sugars, and less than 0.5% of the assimilated label was recovered in the incubation medium. These and previous results (Turgeon and Gowan, 1991, Plant Physiol. 94, 1244–1249) indicate that the phloem loading pathway in Coleus is probably symplastic.Abbreviations 3-OMG 3-O-methyl glucose - PCMBS p-chloromercuribenzenesulfonic acid - SE-CCC sieve-element-companion-cell complex This research was supported by National Science Foundation Grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds.     

Differential sensitivities to glucose and galactose repression of gluconeogenic and respiratory enzymes from Saccharomyces cerevisiae

The synthesis of isocitrate lyase was induced by the presence of ethanol in the chemostat reaching a specific activity of 200 mU·mg^-1 at this induced state. In glucoselimited, derepressed cells, 20 mU·mg^-1 were detected and under repressed conditions isocitrate lyase activity was not detected.The sensitivity of gluconeogenic enzymes: cytoplasmic malate dehydrogenase; fructose 1,6-bisphosphatase and isocitrate lyase as well as the mitochondrial enzymes NADH dehydrogenase and succinate cytochrome c oxidase to glucose and galactose repression were studied in chemostat cultures. Our results show that galactose was less effective as a repressor than glucose. Malate dehydrogenase was completely inactivated by glucose, whereas galactose only produced a 78% decrease of specific activity. Fructose 1,6-bisphosphatase and isocitrate lyase were completely inactivated by both sugars but at different rate. Glucose produced an 85% decrease of specific activity of the mitochondrial enzymes whereas galactose only decrease an 67%.     

Soluble carbohydrates of Pinus sylvestris L. sapwood and heartwood

Summary The amounts of glucose, fructose, sucrose, arabinose/galactose, raffinose/stachyose and starch were investigated in the outer sapwood, innermost sapwood, transition zone and heartwood of four stems of Pinus sylvestris L. The samples were taken in October and the determination of the compounds was done enzymatically. It was not possible to distinguish arabinose from galactose and raffinose from stachyose. The amounts of glucose, fructose and sucrose were greatest in the outer sapwood and decreased gradually towards the innermost sapwood and the heartwood. In the outermost heartwood glucose, fructose and sucrose were only present in trace amounts. Raffinose/stachyose showed highest concentrations in the outer sapwood and decreased towards the heartwood. In contrast, the concentrations of arabinose/galactose increased towards the heartwood and the greatest amount was found in the inner heartwood. When identified by thin-layer chromatography (TLC), arabinose was found to be present in greater amounts than galactose. The amount of starch decreased markedly towards heartwood. However, the amounts of sugars in all the studied stems was very variable. The changes in the amounts of carbohydrates in the different zones of the stems and the possible relationships of these phenomena with heartwood formation are discussed.     

Sugar Transport and Metal Binding in Yeast

The uptake of sugars by yeast can be separated into two classes. The first involves the uptake of sorbose or galactose by starved cells, and the uptake of glucose by iodoacetate-poisoned cells. These uptakes do not involve any changes in Ni⁺⁺- or Co⁺⁺-binding by the cell surface, are not inhibited by Ni⁺⁺, are inhibited by UO₂ ⁺⁺ in relatively high concentrations, are characterized by high Michaelis constants and low maximal rates and by a final equilibrium distribution of the sugars. The second involves the uptake of glucose in unpoisoned cells and galactose in induced cells. These uptakes are characterized by a reduction of Ni⁺⁺- and Co⁺⁺-binding, by a partial inhibition by Ni⁺⁺, by an inhibition with UO₂ ⁺⁺ in relatively low concentrations, and by a low Km and a high Vm. In the case of galactose in induced cells, previous studies demonstrate that the sugar is accumulated against a concentration gradient. It is suggested that the first class of uptakes involves a "facilitated diffusion" via a relatively non-specific carrier system, but the second represents an "uphill" transport involving the highly specific carriers, and phosphoryl groups (cation-binding sites) of the outer surface of the cell membrane.     

Transport and Metabolism of Lactose, Glucose, and Galactose in Homofermentative Lactobacilli

A number of species of lactobacilli were examined for their ability to ferment both the glucose and galactose moieties of lactose. Lactobacillus helveticus strains metabolized both the glucose and galactose moieties, whereas L. bulgaricus, L. lactis, and L. acidophilus strains metabolized only the glucose moiety and released galactose into the growth medium. All four species tested contained β-galactosidase activity, and no significant phospho-β-galactosidase activity was observed. L. bulgaricus and L. helveticus had a phosphoenolpyruvate (PEP):glucose phosphotransferase system for the uptake of glucose, but no evidence for a PEP:lactose phosphotransferase or PEP:galactose phosphotransferase system was obtained.     

The metabolism of galactose and the raffinose oligosaccharides by germinating bambarra groundnut seeds

Stachyose is present in the highest amount in the soluble sugar fraction of dry bambarra groundnut cotyledons, followed in descending order by raffinose, sucrose and verbascose. During germination in the dark, the stachyose and raffinose content decrease rapidly, but there is little change in the relatively small amount of verbascose present. The sucrose content increases rapidly during the first two weeks and decreases thereafter. Free glucose and fructose were present in the cotyledons after the 7th day and gradually increased in amount with time of germination. Free galactose and other galactose-containing oligosaccharides were not detected in either the dry or germinated bambarra seeds. During germination, galactose was the only identifiable sugar, aside from traces of sucrose, glucose and fructose, in the extracted soluble sugar fraction in the embryonic axes of all ages when the tissue was incubated with D-[1¹⁴C] galactose. With the cotyledons, however, most of the radioactivity was in glucose and fructose during the early period of germination and in sucrose later. A small fraction of the radioactivity was lost as CO₂.     

Carbon Source Requirements for Exopolysaccharide Production by Lactobacillus casei CG11 and Partial Structure Analysis of the Polymer

Exopolysaccharide production by Lactobacillus casei CG11 was studied in basal minimum medium containing various carbon sources (galactose, glucose, lactose, sucrose, maltose, melibiose) at concentrations of 2, 5, 10, and 20 g/liter. L. casei CG11 produced exopolysaccharides in basal minimum medium containing each of the sugars tested; lactose and galactose were the poorest carbon sources, and glucose was by far the most efficient carbon source. Sugar concentrations had a marked effect on polymer yield. Plasmid-cured Muc^- derivatives grew better in the presence of glucose and attained slightly higher populations than the wild-type strain. The values obtained with lactose were considerably lower for both growth and exopolysaccharide yield. The level of specific polymer production per cell obtained with glucose was distinctively lower for Muc^- derivatives than for the Muc⁺ strain. The polymer produced by L. casei CG11 in the presence of glucose was different from that formed in the presence of lactose. The polysaccharide produced by L. casei CG11 in basal minimum medium containing 20 g of glucose per liter had an intrinsic viscosity of 1.13 dl/g. It was rich in glucose (76%), which was present mostly as 2- or 3-linked residues along with some 2,3 doubly substituted glucose units, and in rhamnose (21%), which was present as 2-linked or terminal rhamnose; traces of mannose and galactose were also present.     

The Effect of Potassium on the Intestinal Transport of Glucose

The rate of absorption of glucose, galactose, and 3-0-methylglucose was studied in the rat's small intestine perfused in situ with isosmotic solutions containing these sugars and Na₂SO₄ or K2SO₄. The presence of high [K⁺] in the lumen enhances absorption of glucose but not that of galactose or of 3-0-methylglucose. The potassium stimulation is apparent at higher glucose concentrations where primarily carrier-mediated diffusion is involved in the translocation. In this case potassium stimulates transport even if it is the only cation in the lumen. The potassium-stimulated intestine produces more glycogen with higher specific activity than the control gut. Lactic acid production by the intestine is markedly enhanced if the intestinal lumen is perfused with a solution containing glucose and high [K⁺]. It is concluded that potassium does not affect permeability or the specific sugar transport system of the gut, but enhances intracellular metabolic disappearance of glucose thereby creating a larger luminal intracellular concentration gradient which in turn enhances the rate of carrier-facilitated entry.     

Fasciola hepatica: A light and electron microscope autoradiographic study of incorporation of monosaccharides into glycogen and glycoprotein

Incorporation of [³H]galactose and [³H]glucose into the parenchyma, tegument, testis, and muscle of Fasciola hepatica slices was studied by lightand electron-microscope autoradiography. “Accumulation” labeling periods of up to 60 min were used.Both monosaccharides were found to be readily incorporated into glycogen in the parenchymal cells and muscle and [³H]glucose entered the glycogen stores of spermatozoa.No evidence was found for the involvement of any particular cell organelle in glycogenesis, but the demonstration of high synthetic activity in parenchymal evaginations to the base of the surface syncytial tegument supports physiological evidence that glucose enters the fluke mainly across the tegument.Ethylene glycol-dehydrated preparations showed that [³H]galactose was incorporated into glycoprotein by Type I tegumental cells, and perhaps also by sperm morulae. The carbohydrate component seems to be added to the tegumental secretions in the vesicular-lamellar region of the Golgi complex.Following the longest periods of incubation, labeling was observed in the tubules connecting the tegumental cells and syncytium, but not in the surface syncytium itself.     

Sugar Transport into Storage Tubers of Stachys sieboldii Miq.: Evidence for Symplastic Unloading and Stachyose Uptake into Storage Vacuoles by an H+-Antiport Mechanism*

Following assimilation of ¹⁴CO₂ by leaves of Stachys sieboldii, ¹⁴C-stachyose is translocated into the tubers. Stachyose is accumulated and stored in the vacuoles of the pith parenchyma. Protoplasts and vacuoles were isolated and the uptake of sugars was examined. Uptake of sucrose and sucrosyl oligosaccharides of the raffinose family by protoplasts was very low compared to glucose. Transport parameters for glucose indicated a carrier mediated transport in the lower concentration range which was superimposed by diffusion at higher concentrations (> 10 mM). The very low sugar uptake by protoplasts and the sparse enzyme activities of stachyose synthase in the storage parenchyma as well as acid invertase and α-galactosidase in the cell walls indicated symplastic unloading of stachyose in the tubers. Experiments on ¹⁴C-stachyose uptake by isolated vacuoles confirmed previous observations by Keller (1992). Isolated vacuoles exhibited ATP and PP hydrolysis and were capable of generating a proton gradient across the tonoplast by a V-type H⁺-ATPase and H⁺-PPase. This was demonstrated by fluorescence quenching of quinacrine. Fluorescence could be restored by the addition of gramicidin and partly recovered by the addition of stachyose; mannitol, sorbitol and glucose had no effect. Fluorescence recovery depended on the concentration of stachyose and revealed saturation kinetics (K_m = 28 mM). Comparable results have been obtained with tonoplast vesicles by Greutert and Keller (1993). Experimental data presented here provide circumstantial evidence for symplastic unloading of stachyose in the tubers of Stachys sieboldii and demonstrate that the stachyose concentration in the cytoplasm of storage parenchyma cells is kept low by active stachyose transport into the vacuoles. The results suggest a stachyose/H⁺-antiport system.     

Anti-glycosyl antibodies: Preparation and characterization of rabbit anti-galactose and anti-lactose antibodies

Anti-galactose and anti-lactose antibodies have been isolated from the antisera of rabbits immunized with non-viable cells of $\text{Streptococcus}$$\text{faecalis}$, strain N containing an antigenic diheteroglycan of glucose and galactose in the cell wall. The anti-galactose antibodies are specific for the galactosyl moiety while the anti-lactose antibodies are specific for the lactosyl moiety of the diheteroglycan. Hapten inhibitions with galactose and lactose, the sedimentation constant, the immunoglobulin type, the carbohydrate content, the electrophoretic mobility and the amino acid composition have been determined for the two new types of anti-glycosyl antibodies.     

Roles of extracellular lactose hydrolysis in cellulase production by Trichoderma reesei Rut C30 using lactose as inducing substrate

Lactose, an inexpensive, soluble substrate, offers reasonably good induction for cellulase production by Trichoderma reesei. The fungus does not uptake lactose directly. Lactose is hydrolyzed to extracellular glucose and galactose for subsequent ingestion. The roles of this extracellular hydrolysis step were investigated in this study. Batch and continuous cultures were grown on the following substrates: lactose, lactose–glycerol mixtures, glucose, galactose, and glucose–galactose mixtures. Cell growth, substrate consumption, lactose hydrolysis, and lactase and cellulase production were followed and modeled. Cells grew much faster on glucose than on galactose, but with comparable cell yields. Glucose (at >0.3 g/L) repressed the galactose consumption. Cellulase synthesis was growth-independent while lactase synthesis was growth-dependent, except at D < ∼0.065 h⁻¹ where a basal level lactase production was observed. For cellulase production the optimal D was 0.055–0.065 h⁻¹ where the enzyme activity and productivity were both near maxima. The model suggested that lactase synthesis was subject to weak galactose repression. As the galactose concentration increased at high D (>0.1 h⁻¹), lactase synthesis became repressed. The insufficient lactase synthesis limited the lactose hydrolysis rate. Extracellular lactose hydrolysis was concluded to be the rate-limiting step for growth of T. reesei Rut C30 on lactose.     

ght2 + is required for UDP-galactose synthesis from extracellular galactose by Schizosaccharomyces pombe

Schizosaccharomyces pombe has eight hexose transporter genes, ght1 ⁺ to ght8 ⁺. Here we report that ght2 ⁺, which is highly expressed in the presence of glucose, is essential for UDP-galactose synthesis from extracellular galactose when cells grow on glucose. The galactosylation defect of a uge1Δ mutant defective in synthesis of UDP-galactose from glucose was suppressed in galactose-containing medium, but disruption of ght2 ⁺ in the uge1Δ mutant reversed suppression of the galactosylation defect. Expression of Saccharomyces cerevisiae GAL2 in uge1Δght2Δ cells suppressed the defective galactosylation phenotype in galactose-containing medium. These results indicate that galactose is transported from the medium to the cytosol in a Ght2-dependent manner, and is then converted into UDP-galactose.     

Structure of the core regions in lipopolysaccharides from Escherichia coli K12 W2252-11U−, the Ter-15 mutant,and Ter-15 (F′-lac) and Ter-15 (F+) cells

From Escherichia coli K12 W2252-11U^? cells, the Ter-15 mutant, the Ter-15 (F′-lac) and the Ter-15 (F⁺) cells, lipopolysaccharides were isolated and the primary structure of its core oligosaccharides was elucidated. When the F′-lac episome is transferred to the Ter-15 mutant by conjugation, the structure of the glucose III(1 → 3)glucose II(1 → 3)glucose I residue and the galactose I(1 → 2)-linked to the glucose I residue in the core oligosaccharide from the Ter-15 mutant changes into the structure of the glucose IV(1 → 6)glucose III(1 → 2)glucose II(1 → 3)glucose I residue and the galactose I (1 → 6)-linked to the glucose I residue in the core oligosaccharide from the Ter-15 (F′-lac) cells, but the core oligosaccharide in the Ter-15 (F⁺) cells is the same structure with that of the core oligosaccharide from the Ter-15 mutant when F⁺ episome is transferred to the Ter-15 mutant. Also, the core oligosaccharide from the Ter-15 (F′-lac) cells shows the same structure with that of the core oligosaccharide from E. coli K12 W2252-11U^? cells (the parent cells). As the result, the ability to produce the structure of the core oligosaccharide in E. coli K12 W2252-11U^? cells is recovered in the Ter-15 (F′-lac) cells by the dominant expression of lac gene or its containing DNA segment in F′-lac episome.     

Author index for volume 88

Age changes in utilization of glucose and galactose were studied in primary cultures of rat hepatocytes. (1) With increasing age of donor from 2 to 10 weeks, the rate of galactose utilization fell while that of glucose utilization increased from a negative value (net production) to a level approximately double that of galactose. Glucose production could account for about 80% of the galactose taken up by cells from 2- to 3-week-old rats. (2) With increasing time of culture there was a fall in galactose utilization and an increase in glucose utilization comparable to the changes with increasing age in vivo. (3) The change in utilization of each sugar was independent of the change in utilization or availability of the other. It is suggested that the switch from galactose to glucose as the preferred substrate in vivo is not determined by dietary availability of these hexoses. (4) The increased utilization of glucose by cells of older animals was associated with a decrease in responsiveness to the glycogenic action of insulin. The change in responsiveness was not due to a decrease in insulin binding capacity of the cells.