The relationship of root-cap slimes to proteins

1. The patterns of incorporation of radioactivity from d-[U-(14)C]glucose into the pectic components of sections of sycamore roots changed so that sections nearer the tip incorporated relatively more label into arabinose and galactose compared with uronic acid. 2. Radioactive maize root-cap slime was prepared and found to contain three water-soluble component polymers which were electrophoretically (i) neutral, (ii) weakly acidic and (iii) strongly acidic at pH6.5. The neutral component was a glucan. The other components, which could be degraded by trans-elimination, consisted of an acidic backbone chain composed of galacturonic acid and glucose, attached to which were different proportions of neutral sugars. Arabinose, galactose and fucose, the main neutral sugars of the weakly and strongly acidic materials, were absent from the neutral fraction. 3. Fucose was a major sugar in maize-root slime and in a slime of similar composition synthesized by a maize callus of shoot origin. Only trace amounts were found in sycamore, pea and wheat root tips, and in pectin prepared from maize roots and coleoptiles. A high proportion of fucose is therefore a chemical characteristic of maize slime, and slime synthesis indicated a state of differentiation of the tissue. 4. The similarity between the slime and pectin is discussed; slime is a form of pectin modified in such a way as to provide a hydrated protective coating around the root tip.     

Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in pine and fir.

A protein fraction [precipitate obtained between 40 and 65% (NH4)2SO4 satn.] prepared from cambial cells, differentiating xylem cells and differentiated xylem cells of pine and fir trees contained all the enzymes required for the nucleoside diphosphate sugar interconversions. By using UDP-D-[U-14C]glucose or UDP-D-[U-14C]galactose, UDP-D-[U-14C-]glucuronic acid and UDP-D-[U-14C]xylose as substrates, the activities of UDP-D-galactose 4-epimerase (DC 5.1.3.2), UDP-D-xylose 4-epimerase(EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D-glucuronate 4-epimerase (EC5.1.3.6), UDP-d-glucuronate decarboxylase (EC 4.1.1.35) were measured at different stages of cell-wall development. The specific activities and the activities per cell of these enzymes varied during differentiation of cambium to xylem according to the type polysaccharide synthesized. Variations were also found between the two species investigated. These data, compared with those obtained in out previous work on angiosperms [see the preceding paper, Dalessandro & Northcote (1977) Biochem. J. 162, 267-279], suggest that some control of polysaccharide synthesis operates at the level of the formation of the precursors of pectin and hemicellulose syntheses.     

Influence of cations at the plasma membrane in controlling polysaccharide secretion from sycamore suspension cells

1. Three soluble polysaccharides and a soluble protein containing hydroxyproline were secreted by sycamore suspension cultures. l-[1-(3)H]Fucose was incorporated solely into the fucose of fucoxyloglucan and l-[1-(14)C]arabinose mainly into the arabinose of arabino-galactan. [U-(14)C]Glucose was a general precursor for soluble protein and polysaccharides. 2. The steady-state rate of secretion of all the polymers was increased within seconds of adding various electrolytes and polyelectrolytes to the growth medium. The increased secretion was induced by cations at the outer surface of the plasma membrane. It was brought about by a stimulation of the normal mechanisms of cell-wall polysaccharide secretion. It was partly inhibited by anaerobiosis or sodium arsenate and was unaffected by temperature changes in the range 0-35 degrees C. 3. The precursor pool from which secretion was induced contained completely synthesized polysaccharides and was probably located in the Golgi-derived vesicles. The results indicated that the endoplasmic reticulum did not secrete polysaccharide directly to the cell exterior. 4. The various cations probably induced secretion by causing a depolarization of the negative electric potential of the cell surface, and this resulted in the fusion of vesicles with the plasma membrane. 5. Analogy with exocytosis and pinocytosis in various animal tissues suggested that the decreased surface potential brought about membrane fusion by causing an increase in plasma-membrane permeability to Ca(2+). 6. The results showed that the fusion of vesicles with the plasma membrane was rate-limiting and a potential control point. Auxin-stimulated cell-wall deposition could be a result of a stimulated influx of Ca(2+) causing vesicle fusion with the plasma membrane.     

Studies on the enzymic N-acylation of amino sugars in the sheep colonic mucosa

1. d-[2-(14)C]Glucose, [2-(14)C]acetate, hydroxy[3-(14)C]pyruvate, [3-(14)C]pyruvate and [U-(14)C]glycine were incorporated by surviving scrapings of sheep colonic mucosal tissue into glycoprotein. 2. d-[2-(14)C]Glucose, [2-(14)C]acetate, incorporated hydroxy-[3-(14)C]pyruvate and [3-(14)C]pyruvate resulted in labelling of each of the monosaccharide residues of the glycoprotein, namely N-glycollylneuraminic acid, N-acetylneuraminic acid, galactose, fucose, glucosamine and galactosamine. [U-(14)C]Glycine was incorporated as glycyl and seryl residues of the glycoprotein. 3. Despite N-glycollylneuraminic acid being quantitatively the predominant sialic acid (N-glycollylneuraminic acid and N-acetylneuraminic acid were 8.5 and 5.2% by weight of the glycoprotein respectively) the corresponding ratio of the radio-active labelling from d-[2-(14)C]glucose in N-glycollylneuraminic acid to that in N-acetylneuraminic acid was 1.00:7.27 (expressed as percentages of the total radioactivity in the glycoprotein). Neutral sugar, hexosamine and N-acetylneuraminic acid residues of the mucoprotein were each labelled to a similar extent. 4. Similarly, the ratio of the radioactivity in N-glycollylneuraminic acid to that in N-acetylneuraminic acid in the mucoprotein from tissue incubations with [2-(14)C]-acetate was 1.0:4.0. 5. Both [2-(14)C]acetate and [2-(14)C]glucose with whole tissue led to labelling of the N-glycollyl substituent and of the main nonose skeleton of the N-glycollylneuraminic acid. In whole-tissue incubations, [3-(14)C]pyruvate was also a precursor of radioactive N-glycollylneuraminic acid. 6. Hydroxy[3-(14)C]-pyruvate and [U-(14)C]glycine caused labelling of the carbohydrate and peptide residues of the glycoprotein, but did not give rise to labelling in the N-glycollylneuraminic acid residues. 7. With a wide variety of possible N-glycollyl precursors (fructose 6-phosphate, hydroxypyruvate, glycollate and chemically synthesized glycollyl-CoA) biosynthesis of N-glycollylglucosamine was not observed in cell-free preparations.     

Biosynthesis of intestinal mucins. Effect of puromycin on mucoprotein biosynthesis by sheep colonic mucosal tissue

1. Surviving sheep colonic mucosal tissue incorporated l-[U-(14)C]threonine when incubated in Krebs medium III at 37 degrees in an atmosphere of oxygen, into a well-characterized mucoprotein fraction, isolated by papain digestion of the incubated scrapings. 2. Acidic hydrolysis and chromatography of the labelled mucoprotein showed that threonine was the only constituent to become labelled. In the presence of puromycin the incorporation of l-[U-(14)C]threonine was considerably diminished (6.7 and 18.5% of control in duplicate experiments). Furthermore, puromycin also decreased incorporation of radioactivity from d-[U-(14)C]-glucose (48.0 and 31.6% of control) and (35)SO(4) (2-) (21.2 and 23.6% of control) into the mucoprotein fraction. 3. In a puromycin-inhibited system, with d-[U-(14)C]-glucose, where the overall specific radioactivity of the mucoprotein was 48% of control, the labelling of the individual monosaccharide constituents (as% of control) was: N-acetylneuraminic acid, 44%; N-glycollylneuraminic acid, 61%; hexosamines, 46%; fucose, 68%; galactose, 34%.     

Characterization of [14C]apiogalacturonans synthesized in a cell-free system from Lemna minor.

Radioactive polysaccharide was synthesized when uridine 5′-(α-d-[U-¹⁴C]apio-d-furanosyl pyrophosphate) (containing some uridine 5′-(α-d-[U-¹⁴C]xylopyranosyl pyrophosphate)) was incubated with a particulate enzyme preparation from Lemna minor. Characterization experiments established that the product: (i) was insoluble in methanol and water, (ii) contained d-[U-¹⁴C]apiose (75%) and d-[U-¹⁴C]xylose (25%), and (iii) was soluble in 1% ammonium oxalate. The material solubilized by ammonium oxalate (solubilized product): (i) was separated into five fractions by column chromatography with diethylaminoethyl-Sephadex (DEAE-Sephadex), (ii) contained [U-¹⁴C]apiobiose side chains that were removed by hydrolysis at pH 4, and (iii) was degraded by fungal pectinase. Both d-[U-¹⁴C]apiose residues of the [U-¹⁴C]apiobiose side chains were synthesized in vivo since radioactivity was distributed equally between the two residues. The presence of uridine 5′-(α-d-galactopyranosyluronic acid pyrophosphate) during synthesis of radioactive polysaccharide resulted in: (i) an increase in the incorporation of radioactive d-[U-¹⁴C]apiose into solubilized product, (ii) an increase in the ratio of d-[U-¹⁴C]apiose to d-[U-¹⁴C]xylose present in solubilized product, (iii) an increase in the amount of [U-¹⁴C]apiobiose plus d-[U-¹⁴C]apiose released from the solubilized product by hydrolysis at pH 4, and (iv) a tighter binding of the solubilized product to DEAE-Sephadex. These results show that apiogalacturonans similar to or the same as those synthesized by the intact plant were synthesized in the particulate enzyme preparation isolated from L. minor. [¹⁴C]Apiogalacturonans completely free of d-[U-^l4C]xylose were not isolated. The [¹⁴C]apiogalacturonan with the least d-[U-¹⁴C]xylose still had 4.8% of its radioactivity present in d-[U-¹⁴C]xylose. The possibility remains that d-xylose is a normal constituent of the apiogalacturonans of the cell wall of L. minor.     

Radioisotope ratios discriminate between competing pathways of cell wall polysaccharide and RNA biosynthesis in living plant cells

Cell wall polysaccharides are synthesized from sugar-nucleotides, e.g. uridine 5'-diphosphoglucose (UDP-Glc), but the metabolic pathways that produce sugar-nucleotides in plants remain controversial. To help distinguish between potentially 'competing' pathways, we have developed a novel dual-radiolabelling strategy that generates a remarkably wide range of 3H:14C ratios among the various proposed precursors. Arabidopsis cell cultures were fed traces of D-[1-(3)H]galactose and a 14C-labelled hexose (e.g. D-[U-14C]fructose) in the presence of an approximately 10(4)-fold excess of non-radioactive carbon source. Six interconvertible 'core intermediates', galactose 1-phosphate <--> UDP-galactose <--> UDP-glucose <--> glucose 1-phosphate <--> glucose 6-phosphate <--> fructose 6-phosphate, showed a large decrease in 3H:14C ratio along this pathway from left to right. The isotope ratio of a polysaccharide-bound sugar residue indicates from which of the six core intermediates its sugar-nucleotide donor substrate stemmed. Polymer-bound galacturonate, xylose, arabinose and apiose residues (all produced via UDP-glucuronate) stemmed from UDP-glucose, not glucose 6-phosphate; therefore, UDP-glucuronate arose predominantly by the action of UDP-glucose dehydrogenase rather than through the postulated competing pathway leading from glucose 6-phosphate via myo-inositol. The data also indicate that UDP-galacturonate was not formed by a hypothetical UDP-galactose dehydrogenase. Polymer-bound mannose and fucose residues stemmed from fructose 6-phosphate, not glucose 1-phosphate; therefore GDP-mannose (guanosine 5'-diphosphomannose) arose predominantly by a pathway involving phosphomannose isomerase (via mannose phosphates) rather than through a postulated competing pathway involving GDP-glucose epimerization. Curiously, the ribose residues of RNA did not stem directly from hexose 6-phosphates, but predominantly from UDP-glucose; an alternative to the textbook pentose-phosphate pathway therefore predominates in plants.     

Stimulation of polysaccharide formation by 2,4-dichlorophenoxyacetic acid in callus tissues of Arabidopsis thaliana

A relatively high concentration of 2,4-dichlorophenoxyacetic acid (45 μ M ) in solid culture medium stimulated the formation and secretion of mucilage polysaccharides by callus tissues of Arabidopsis thaliana L. Heynh. (line Estland). The mucilage was composed of at least two polysaccharides as revealed by gel chromatography on Sepharose 4B: the major component (87%) eluted in the void volume (molecular weight 2 × 10⁶ or greater) and the minor component (13%) eluted in the molecular weight range from 2 × 10⁴ to 4 × 10⁵. Both polysaccharide components contained small amounts of uronic acids. The major polysaccharide consisted mostly of galactose (49%), arabinose (28%) and fucose (10%), whereas the minor one consisted of galactose (44%), xylose (18%), arabinose (14%) and rhamnose (14%). One of the components of the secreted mucilage seems to be an arabinogalactan.     

Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: II. Evidence for the Participation of Uridine Diphosphoxylose and Free Xylose as Intermediates

myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-(3)H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-(14)C]inositol, myo-[2-(3)H]inositol, l-[5-(14)C]arabinose, and d-[5R,5S-(3)H]xylose.myo-[2-(14)C]Inositol and l-[5-(14)C]arabinose produced labeled glucose with similar patterns of distribution of (14)C, 35% in C1, and 55% in C6. Arabinosyl units were labeled exclusively in C5. Incorporation of (3)H into arabinosyl and xylosyl units in pollen labeled with myo-[2-(3)H]inositol was repressed when unlabeled l-arabinose was included in the germination medium and a related (3)H exchange with water was stimulated. Results are consistent with a process of glucogenesis in which the myo-inositol oxidation pathway furnishes UDP-d-xylose as a key intermediate for conversion to hexose via free d-xylose and the pentose phosphate pathway.Additional evidence for this process was obtained from pollen labeled with d-[5R,5S-(3)H]xylose or myo-[2-(3)H]inositol which produces d-[5R-(3)H]xylose. Glucosyl units from polysaccharides in the former had 11% of the (3)H in C1 and 78% in C6 while glucosyl units in the latter had only 4% in C1 and 78% in C6. Stereochemical considerations involving selective exchange with water of prochiral-R (3)H in C1 of fructose-6-P during conversion to glucose provide explanation for observed differences in the metabolism of these 5-labeled xyloses.Incorporation of (3)H from myo-[2-(3)H]inositol into arabinosyl and xylosyl units of pollen polysaccharides was unaffected by the presence of unlabeled d-xylose in the medium. Exchange of (3)H with water was greatly affected, decreasing from a value of 21% exchange in the absence of unlabeled d-xylose to 5% in the presence of 6.7 mmd-xylose.d-Xylose was rapidly utilized for glucogenesis by germinated pollen tubes. This observation supports the view that free d-xylose is an important intermediate following breakdown of UDP-d-xylose during myo-inositol-linked glucogenesis.     

Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Three-day-cultured cells of Vinca rosea L. (in the cell division phase) and 5-day-cultured cells (in the cell expansion phase) prelabelled with d -[U-¹⁴C] glucose were incubated in a medium containing unlabelled glucose. After various periods of chase, extra-cellular polysaccharides (ECP) and cell walls were isolated, and cell walls were fractionated into pectic substances, hemicellulose, and cellulose fractions. After acid hydrolysis, the radioactive constituents in the pectic substances and hemicellulose fractions were analyzed. Active turnover was observed in arabinose and galactose in the hemicellulose fraction of cell walls, while the constituents of the pectic substances, and xylose and glucose in the hemicellulose fraction did not undergo active turnover. The proportion of radioactivities of arabinose and galactose in total radioactivity of ECP increased markedly after chasing. These results indicate that arabinogalactan was synthesized, deposited in the cell wall, degraded rapidly, and made soluble in the medium as a part of ECP.     

Partial structure of a polysaccharide from leaves of Welwitschia mirabilis

Gum-tears from the leaves of Welwitschia mirabilis contain a polysaccharide composed of arabinose, galactose and glucuronic acid as main constituents with xylose, fucose and rhamnose in smaller quantities. Periodate oxidation and permethylation studies indicated that the gum could consist of a framework of glucuronic acid residues linked 1 → 4 and galactose residues linked 1 → 6 and of short chains of arabinose, xylose, fucose and rhamnose linked 1 → 3 to both residues. All rhamnose and fucose and part of arabinose were found as non-reducing terminal units.     

Extracellular polysaccharides of some basidiomycetes.

Culture filtrates of four basidiomycete fungi, Stereum strigoso-zonatum, Fomes australis, Trametes lilacinogilva and Polyporus tumulosus were fractionated and examined for polysaccharide content. Acid hydrolysis showed the presence of galactose, mannose, xylose, fucose and glucose. Their relative amounts were estimated by gas chromatography of the corresponding alditol acetates. Galactose and mannose were the major constituent sugars, amounting to more than 50% of the total. One of the polysaccharides, a fucogalactomannan elaborated by P. tumulosus, was isolated in a purified form. It was shown to have [alpha]D +42 degrees and contained galactose, mannose, fucose and xylose in the relative proportions 2 : 1 : 1 : 0-2.     

Biosynthesis of myo-inositol and its role as a precursor of cell-wall polysaccharides in suspension cultures of wild-carrot cells

The biosynthesis of myo-inositol (MI) and its role as a precursor of cell-wall polysaccharides was studied in supension cultures of wild carrot (Daucus carota L.) cells. Suspension cultures, grown in the presence or absence of 2,4-dichlorophenoxyacetic acid for 7 and 14d were incubated with [U-¹⁴C]glucose and [2-³H]MI in the presence of different concentrations of unlabeled MI. Synthesis of [¹⁴C]MI from [U-¹⁴C]glucose occurred under all conditions. The amount of MI synthesized from glucose was sharply reduced when 10 mM MI was provided in the medium. Substantial quantities of ³H were incorporated in arabinose, xylose and galacturonic acid isolated and purified from the cell-wall polysaccharides of the cell cultures in various stages of growth or embryogenesis. No ³H was present in the glucose or galactose units of cell-wall polysaccharides. At the four stages of growth and states of development of the carrot cultures used, the MI oxidation pathway contributed to the synthesis of pentosyl and galacturonosyl units of the cell wall. However, the data indicate that the contribution of the MI oxidation pathway to pentosyl and galacturonosyl units is small.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MI myo-inositol     

Characterization and antioxidant activity of Ginkgo biloba exocarp polysaccharides

Ginkgo biloba exocarp polysaccharide (GBEP) was obtained by hot water extraction, the crude polysaccharide was deproteinized by Sevag method and fractionized by a DEAE Sepharose fast flow anion-exchange column. Five fragments were obtained, including neutral polysaccharide (GBEP-N) and four acidic polysaccharides (GBEP-A1, GBEP-A2, GBEP-A3 and GBEP-A4). GBEP-N and GBEP-A3 were further purified by Superdex 200 gel column chromatography. The resulted two fractions GBEP-NN, and GBEP-AA were characterized by FT-IR, and HPGFC (high pressure gel filtration chromatography). Monosaccharide composition was determined by RP-HPLC method of precolumn derivatization with 1-phenyl-3-5-pyrazolone. GBEP-NN was mainly composed of rhamnose, arabinose, mannose, glucose and galactose, while GBEP-AA was mainly made up of mannose, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose. The crude GBEP exhibited certain antioxidant activity. At the concentration of 5 mg/mL, the hydroxyl radical scavenging effect of GBEP was 90.52%, greater than 77.37% for the positive control ascorbic acid.     

An Examination of Centrifugation as a Method of Extracting an Extracellular Solution from Peas, and Its Use for the Study of Indoleacetic Acid-induced Growth

A technique of centrifuging pea epicotyl sections which extracts water-soluble cell wall polysaccharides with less than 1.5% cytoplasmic contamination as revealed by malate dehydrogenase activity determinations was developed. Tests for protein, hexose, pentose, and malate dehydrogenase indicate that significant damage to the cells occurs above 3,000g. Below this force, there is little damage, as evidenced by the similar growth rates of centrifuged and noncentrifuged sections. Centrifugation at 1,000g extracts polysaccharides containing rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose. An increase in xylose and glucose, presumably xyloglucan, is induced by treating sections with indoleacetic acid. Much of the alcohol-insoluble, water-soluble polysaccharide within the wall is extractable by centrifugation, since nearly as much arabinose and xylose are extractable by centrifugation as by homogenization. The utility of this method for the study of cell wall metabolism is discussed.     

Changes in cell-wall polysaccharides in relation to seedling development and the mobilisation of reserves in the cotyledons of Lupinus angustifolius cv. Unicrop

Some 22% of the dry weight of the cotyledons of resting seeds of Lupinus angustifolius cv. Unicrop has been shown to be non-starch polysaccharide material comprising the massively thickened walls of the storage mesophyll cells. On hydrolysis this material released galactose (76%), arabinose (13%), xylose (4%), uronic acid (7%): only traces of glucose were detected indicating the virtual absence of cellulose from the walls. Changes in the amount and composition of this material following germination have been studied in relation to parameters of seedling development and the mobilisation of protein, lipid and oligosaccharide reserves. Starch, which was not present in the resting seed, appeared transitorily following germination: under conditions of continuous darkness starch levels were reduced. During the period of bulk-reserve mobilisation, 92% of the non-starch polysaccharide material disappeared from the cotyledons. The residual cell-wall material released galactose (14%), arabinose (19%), xylose (24%) and uronic acid (43%). The galactose and arabinose residues of the cotyledonary cell walls clearly constitute a major storage material, quantitatively as important as protein. The overall role of the wall polysaccharides in seedling development is discussed.     

Biosynthesis of xyloglucan in suspension-cultured soybean cells. Occurrence and some properties of xyloglucan 4-beta-D-glucosyltransferase and 6-alpha-D-xylosyltransferase

A particulate enzyme fraction that catalyzes the transfer of glucose from UDP-[14C]glucose and of xylose from UDP-[14C]xylose into a xyloglucan has been isolated from suspension-cultured soybean cells. The incorporation of radioactivity from [14C]xylose into the polysaccharide was dependent on the presence of UDP-glucose in the incubation mixture, and that from [14C]glucose was dependent on the concentration of UDP-xylose in the mixture. Mn2+ was required for the incorporation of xylose and the optimum concentration of Mn2+ was about 10 mM. This reaction showed a pH optimum at 6.5 to 7.0 in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and was inhibited by phosphate buffer and Tris buffer. On hydrolysis with Trichoderma endoglucanase, the polysaccharide synthesized in vitro gave a pentasaccharide, a hepatasaccharide, and a small amount of non-asaccharide. Based on the results from fragmentation and methylation analyses, the following structures were proposed for the penta- and the heptasaccharides from the xyloglucan synthesized in vitro: (formula, see text).     

Golgi Apparatus Mediated Polysaccharide Secretion by Outer Root Cap Cells of Zea mays. III. Control by Exogenous Sugars

Sugars supplied to germinating seedlings of maize (Zea mays L.) regulate the secretion of polysaccharides by the outer cells of the root cap. The polysaccharide secreted by these cells adheres to the root tip as a droplet and the size of the droplet was used to quantitate polysaccharide secretion. The polysaccharide contains glucose, galacrose, and galacturonic acid residues with smaller quantities of mannose, arabinose, xylose, fucose and rhamnose. These sugars supplied to maize seedlings had marked effects on the rate of polysaccharide secretion by root tips. The effects on secretion were independent of the growth rates of the roots. Glucose, fucose and xylose increased droplet size 1.5–2 fold (as did sucrose, maltose, lacrose, fructose and ribose) whereas galactose, arabinose and galacturonic acid were inhibitory. Mannose increased dropler size 5–7 fold. The marked effect of mannose on polysaccharide secretion was due to an increased rate of secretion combined with a longer phase of extrusion of polysaccharide into the forming droplet. The effect of mannose was partially reversed by inorganic phosphate and other sugars (except for fucose which had no effect or promoted secretion in the presence of mannose). In contrast to sucrose, mannose stimulated secretion in a maize variety having a high sugar endosperm (high endogenous sugar). The results suggest that regulation of secretion by mannose is due to an alteration of normal sugar metabolism; whereas stimulation of secretion by sucrose and other sugars may be due to an increased availability of sugars for metabolism.     

Reactive oxygen species and nitric oxide affect cell wall metabolism in tobacco BY-2 cells

The effects of hydrogen peroxide (H2O2), nitric oxide (NO), and a combination of both on the metabolism of cell wall polysaccharides were studied in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY-2) suspension cultured cells in the presence of D-[U-14C]glucose or D-[U-14C]galactose as radioactive tracers. We found that the radiolabelling of newly synthesised total cell wall polysaccharides (pectins, hemicelluloses and alpha-cellulose), buffer-soluble polysaccharides, and membrane-associated polysaccharides decreased under the influence of exogenous systems generating H2O2 and NO. However, when the total amount of newly synthesised cell wall polysaccharides was calculated as a percentage of the total cellular radioactivity (ethanol-soluble pool plus the homogenate of ethanol-insoluble material), all treatments showed negligible effects in the presence of D-[U-14C]glucose or D-[U-14C]galactose as tracers. This occurred because the treatments generating H2O2, NO and H2O2 plus NO caused a marked decrease in the concentration of the ethanol-soluble pool as well as in the total radioactivity found in the homogenate of the ethanol-insoluble material. Most of the radioactivity taken up by the cells was evolved as 14CO2 during the respiratory processes. A qualitative and quantitative characterisation of the ethanol-soluble pool showed that radioactive UDP-sugars in BY-2 suspension cultured cells were differentially reduced by all treatments. Therefore, the decrease of the newly synthesised cell wall polysaccharides seems to be strictly dependent on the reduction of the UDP-sugars pool.     

The biosynthesis of intestinal mucins. The effect of salicylate on glycoprotein biosynthesis by sheep colonic and human gastric mucosal tissues in vitro

1. Incubation of sheep colonic mucosal scrapings in Krebs-Ringer buffer for 2(1/2)hr. in the presence of salicylate (15mm) resulted in decreased incorporation of radioactivity into the epithelial glycoprotein from the following labelled precursors: 16.6mum-d-[2-(14)C]glucose (83.9% inhibition), 20mum-l-[U-(14)C]threonine (82%) and (35)SO(4) (2-)(79%). Oxygen uptake measured simultaneously was diminished to 41% of the control value. 2. At lower concentrations of salicylate (e.g. 3.75mm), incorporation of 20mum-l-[U-(14)C]threonine was little affected (3-6% inhibition), whereas utilization of 4mum-d-[U-(14)C]glucose and (35)SO(4) (2-) was inhibited (41-48% and 40-59% of the control values respectively). 3. Analysis of the papain-digested glycoprotein from tissue incubations with 16.6mum-d-[2-(14)C]glucose in the presence of salicylate (3.75mm) showed large decreases in labelling of N-acetylneuraminic acid and N-glycollylneuraminic acid residues (57% and 34% of the control values respectively) and of hexosamine constituents (glucosamine, 55% inhibition; galactosamine, 33% inhibition). Labelling of neutral sugars (galactose and fucose) was relatively little affected (9 and 11% inhibition respectively). 4. Glucose 6-phosphate transaminase and glucosamine 6-phosphate acetylase in particle-free enzyme preparations of the sheep tissue were unaffected by salicylate at the above concentrations. Acetyl-CoA synthetase was markedly inhibited. 5. Human gastric mucosa (from operation), on incubation as above, had in one experiment an oxygen consumption of 9.9mul./hr./mg. dry wt. of tissue and incorporated 5mum-d-[U-(14)C]glucose (15.8% of the total radioactivity added) into bound hexosamine (20.6% of the total radioactivity incorporated), hexoses (glucose and galactose, 5.7%) and fucose (14.2%). The presence of salicylate (15mm) decreased the incorporation of 5mum-d-[U-(14)C]glucose into the glycoprotein by 74%, all sugar constituents being affected, without influence on the rate of oxygen consumption. 6. The results suggest an inhibitory effect of salicylate on glycoprotein biosynthesis at the level of the amino sugar intermediates.