Synthesis, characterization and properties of uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)

1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.     

Solubilization and characterization of a galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan

Polygalacturonate 4-α-galacturonosyltransferase (PGA-GalAT), the glycosyltransferase that synthesizes the plant cell wall pectic polysaccharide homogalacturonan, has previously been identified and partially characterized in tobacco membranes. Membrane bound PGA-GalAT catalyzes the transfer of galacturonic acid from UDP-galacturonic acid (UDP-GalA) onto an endogenous acceptor to produce polymeric homogalacturonan ( Doong et al. (1995) Plant Physiol. 109, 141 –152). It is shown here that a galacturonosyltransferase is solubilized from tobacco membranes with a HEPES buffer, pH 6.8, containing 40 mM CHAPS and 2 mM EDTA. The solubilized galacturonosyltransferase was identified as putative PGA-GalAT because it transfered [¹⁴C]GalA from UDP-[¹⁴C]GalA onto exogenous homogalacturonan acceptors with degrees of polymerization (DP) of ≥ 10. Maximal solubilized PGA-GalAT activity in the presence of 0.9 μM UDP-[¹⁴C]GalA required approximately 125 μM exogenous homogalacturonan acceptor [e.g. oligogalacturonide (OGA) of DP 15]. Solubilized PGA-GalAT was active over a broad pH range of 6.3–7.8, and had an apparent K_m for UDP-GalA of 37 μM and a V_max of 290 pmol min^–1 mg^–1 protein. Approximately 44% of the PGA-GalAT activity in detergent-dispersed membranes, corresponding to 21% of the PGA-GalAT activity in intact membranes, was solubilized. PGA-GalAT solubilized with 40 mM CHAPS was shown, by exopolygalacturonase treatment in combination with size exclusion and high performance anion exchange chromatographies, to add a single α-1,4-linked galacturonic acid residue onto an OGA exogenous acceptor of DP 15 to yield an OGA product of DP 16. The significance of the apparent lack of processivity of the solubilized PGA-GalAT is discussed.     

Galactosyltransferase activities in mitochondrial outer membrane: biosynthesis of dolichylmonophosphate-galactose

Mitochondrial outer membranes were prepared from mouse liver homogenates by swelling purified mitochondria in phosphate buffer and were purified on a discontinuous sucrose gradient. Assays for marker enzymes and controls in electron microscopy confirmed the purity and homogeneity of this subfraction. Mitochondrial outer membranes had significant galactosyltransferase activity when incubated with UDP-[14C]galactose: 14C-labelling was found in products extractable with organic solvents and in a residual precipitate. Addition of exogenous dolichylmonophosphate loaded into phosphatidylcholine liposomes strongly enhanced the incorporation of [14C]galactose into chloroform/methanol (2:1, v/v) -extractable products. Thin-layer chromatography of these 2:1 extracts showed that the increase of [14C]galactose incorporation was attributable to the synthesis of a new galactosylated lipid, 'lipid L'. This 'lipid L' has been purified on silicic acid columns by elution with chloroform/methanol (1:1, v/v). The purified 'lipid L' was labile in acid and released [14C]galactose. It had the same chromatographic behaviour as dolichylmonophosphate-mannose in neutral, acid and alkaline solvent systems. Upon incubation in presence of [3H]dolichylmonophosphate and UDP-[14C]galactose, purified 'lipid L' contained both 3H- and 14C-labelling. 'Lipid L', synthesized by mitochondrial outer membranes, was therefore characterized as dolichylmonophosphate-galactose.     

Novel O-D-galacturonoyl esters in the pectic polysaccharides of suspension-cultured plant cells.

Driselase digestion of uronate-6-14C-labeled primary walls of cultured spinach (Spinacia oleracea L.) cells yielded about 18 novel uronate-containing compounds, most of which could be hydrolyzed by cold dilute alkali to yield oligo-[14C]galacturonides. One typical Driselase digestion product (compound 17) yielded alpha-(1-->4)-D-[14C]galacturonotriose(GalA3) upon very mild treatment with alkali (50% yield of GalA3 in 7.2 min at pH 11 and 25 degrees C). One of the three galacturonate residues in compound 17 was reducible to a galactose residue with sodium borohydride, indicating that that GalA residue was esterified, via its--COOH group, to a putative alcohol. Compound 17 had a higher mobility than GalA3 on paper chromatography, indicating that the putative alcohol was relatively nonpolar. The putative alcohol could not have been methanol because Driselase readily hydrolyzed mono-, di-, and trimethyl esters of GalA3 to yield free galacturonic acid. Another Driselase digestion product (compound 12) was a derivative of GalA3 that apparently possessed two nonpolar esterified substituents: one about as labile as in compound 17, and the other approximately 10 times more stable. Compounds 12 and 17 could not labeled by in vivo feeding of [U-14C]cinnamate, suggesting that they were not phenolic conjugates. Similar but chromatographically distinguishable uronate-14C-labeled esters were obtained by Driselase digestion of walls of cultured carrot (Daucus carota L.), Paul's Scarlet rose (Rosa sp.), and tall fescue (Festuca arundinacea Schreber) cells. In spinach, the novel compounds constituted about 5% of the total galacturonate residues of the cell wall. The observations suggest that pectic polysaccharides are linked, via O-D-galacturonoyl ester bonds, to relatively hydrophobic constituents of the primary cell wall. Their possible role in wall architecture is discussed.     

Glucuronosyl-N-acetylglucosaminyl pyrophosphoryldolichol. Formation in SV40-transformed human lung fibroblasts and biosynthesis in rat lung microsomal preparations.

Incubation of SV40-transformed human lung fibroblasts with [3H]glucosamine for 1 h. followed by chloroform:methanol extraction and thin layer chromatographic analysis, revealed the presence of a major radioactive lipid that was isolated and characterized as GIcUA-(1 leads to 4)-GlcNAc-P-P-dolichol. An identical lipid was formed in smaller quantities under similar incubation conditions in several fibroblastic lines, HeLa cells, and in mouse L cells. Rat lung microsomal preparations catalyze the synthesis of the disaccharide lipid in the following sequence of reactions: UDP-[3H]GlcNAc + dolichol-P leads to [3H]GlcNAc-P-P-dolichol (1) [3H]GlcNAc-P-P-dolichol + UDP-[14C]GlcUA leads to [14C]GlcUA-[3H]GlcNAc-P-P-dolichol (2) The double-labeled lipid was identical to the lipid isolated from SV40-transformed fibroblasts with regard to its behavior on thin layer and silicic acid chromatography. Further, the double-labeled disaccharide released from the lipid by mild acid hydrolysis was identical to GlcUA-(1 leads to 4)-GlcNAc in its chromatographic and electrophoretic behavior and in its composition. The occurrence of a polyprenol derivative of GlcUA-(1 leads to 4)-GlcNAc suggests a possible role for this lipid in the biosynthesis of the repeating disaccharide units of proteoglycans, such as heparin.     

Cellulose synthesis by extracts of Acanthamoeba castellanii during encystment. Stimulation of the incorporation of radioactivity from UDP-(14C)glucose into alkali-soluble and insoluble beta-glucans by glucose 6-phosphate and related compounds.

1. The activity of a particulate enzyme prepared from encysting cells of Acanthamoeba castellanii (Neff), previously shown to catalyze the incorporation of glucose from UDP-[14C]glucose into both alkali-soluble and alkali-insoluble beta-(1 leads to 4) glucans, was stimulated several fold by glucose-6-phosphate and several related compounds. 2. Incorporation was observed when [14C]glucose-6-P was incubated with the particles in the presence of UDP-glucose. The results of product analysis by partial acid hydrolysis indicated that glucose-6-P stimulates the formation of both alkali-soluble and alkali-insoluble beta-(1 leads to 4) glucans from UDP-[14C]glucose and was itself incorporated into an alkali-insoluble beta-(1 leads to 4)glucan. 3. When particles incubated with UDP-[14C]glucose and glucose-6-P were reisolated and then reincubated with unlabeled UDP-glucose and glucose-6-P, a loss of counts from the alkali-soluble fraction was detected along with a corresponding rise in the radioactivity of the alkali-insoluble fraction. This suggests that the alkali-soluble beta-glucan was converted to an alkali-insoluble product and possibly may be an intermediate stage in cellulose synthesis.     

Biosynthesis of chondroitin sulfate. Chain termination

Incubation of chick embryo epiphyseal microsomal preparations with either UDP-[14C]GlcUA or UDP-[14C]-GalNAc plus exogenous chondroitin 6-sulfate resulted in the incorporation of either a single [14C]GlcUA or a [14C]GalNAc onto the nonreducing ends of the exogenous glycosaminoglycan. Degradation by chondroitinase ABC yielded the terminal products [14C]Di-OS, [14C]Di-6S, and [14C]GalNAc. Incubations of the microsomal preparations with either UDP-[14C]GlcUA or UDP-GalN[3H]Ac without exogenous chondroitin 6-sulfate resulted in the addition of a single sugar onto the nonreducing end of endogenous chondroitin sulfate. Degradation by chondroitinase ABC yielded the terminal products [14C]Di-OS, [14C]Di-6S, and GalN[3H]Ac in a molar ratio of approximately 1:1:3.5. Incubations of the microsomal preparations with both UDP-[14C]-GlcUA and UDP-GalN[3H]Ac together resulted in formation of [14C,3H]chondroitin chains added to the endogenous chondroitin sulfate. Degradation by chondroitinase ABC resulted in products with a molar ratio of [14C,3H]Di-OS to GalN[3H]Ac varying from approximately 1:1.5 to 1:3. The results of these experiments indicate that chondroitin 6-sulfate terminates at its nonreducing end in a mixture of GlcUA and GalNAc (some sulfated). GalNAc is somewhat more frequent as the terminal sugar and adds more readily to endogenous acceptors.     

Biosynthesis of heparin. O-sulfation of D-glucuronic acid units

Incubation of a microsomal fraction from murine mastocytoma, with UDP-[1-3H]GlcA, UDP-GlcNAc, and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), yielded labeled, N-sulfated polysaccharides, in which most of the incorporated O-sulfate groups were located at C2 of L-iduronic acid and at C6 of D-glucosamine units. Analysis by anion-exchange high pressure liquid chromatography of disaccharides, generated by deaminative cleavage of these polysaccharides, revealed that, in addition, an appreciable portion of the -GlcNSO3-HexA-GlcNSO3- sequences in the intact polymers contained O-sulfated (at C2 or C3) D-glucuronic acid units. Calculations based on such compositional analysis of the N- and O-sulfated biosynthetic product, isolated by chromatography on DEAE-cellulose, showed that glucuronosyl 2/3-O-sulfate accounted for approximately 12% of the total incorporated O-sulfate groups. With [35S]PAPS (at a low total PAPS concentration) as an alternative source of label, the sulfated glucuronic acid residues were again detectable, albeit in much smaller amounts (1.8% of the total O-sulfate groups). Incorporation of label from UDP-[5-3H]GlcA was retained by the O-sulfated glucuronic acid units, thus demonstrating that these components had in fact been formed by sulfation of glucuronic acid residues and not by "back epimerization" of sulfated iduronic acid units. Structural analysis of polysaccharide intermediates at various stages of biosynthetic polymer modification, separated by ion-exchange chromatography, showed O-sulfation of glucuronic and iduronic acid units to appear simultaneously and before the 6-O-sulfation of glucosamine residues.     

In vitro synthesis and O acetylation of peptidoglycan by permeabilized cells of Proteus mirabilis.

The synthesis and O acetylation in vitro of peptidoglycan by Proteus mirabilis was studied in microorganisms made permeable to specifically radiolabelled nucleotide precursors by treatment with either diethyl ether or toluene. Optimum synthesis occurred with cells permeabilized by 1% (vol/vol) toluene in 30 mM MgCl2 in in vitro experiments with 50 mM Tris-HCl buffer (pH 6.80). Acetate recovered by mild base hydrolysis from sodium dodecyl sulfate-insoluble peptidoglycan synthesized in the presence of UDP-[acetyl-1-14C]N-acetyl-D-glucosamine was found to be radioactive. Radioactivity was not retained by peptidoglycan synthesized when UDP-[acetyl-1-14C]N-acetyl-D-glucosamine was replaced with both unlabelled nucleotide and either [acetyl-3H]N-acetyl-D-glucosamine or [glucosamine-1,6-3H]N-acetyl-D-glucosamine. In addition, no radioactive acetate was detected in the mild base hydrolysates of peptidoglycan synthesized in vitro with UDP-[glucosamine-6-3H]N-acetyl-D-glucosamine as the radiolabel. Chasing UDP-[acetyl-1-14C]N-acetyl-D-glucosamine with unlabelled material served to increase the yield of O-linked [14C]acetate, whereas penicillin G blocked both peptidoglycan synthesis and [14C]acetate transfer. These results support the hypothesis that the base-labile O-linked acetate is derived directly from N-acetylglucosamine incorporated into insoluble peptidoglycan via N----O transacetylation and not from the catabolism of the supplemented peptidoglycan precursors followed by subsequent reactivation of acetate.     

Biosynthesis of pentosyl lipids by pea membranes.

Pea membranes were incubated with UDP-[14C]xylose or UDP-[14C]arabinose and sequentially extracted with chloroform/methanol/water (10:10:3, by vol.) and sodium dodecyl sulphate (2%, w/v). An active epimerase in the membranes rapidly interconverted the two pentosyl nucleotides. Chromatographic analysis of the lipid extract revealed that both substrates gave rise to xylose- and arabinose-containing neutral lipids, xylolipid with properties similar to a polyisoprenol monophosphoryl derivative, and highly charged lipid-linked arabinosyl oligosaccharide. When UDP-[14C]pentose or the extracted lipid-linked [14C]arabinosyl oligosaccharide were used as substrates, their 14C was also incorporating into sodium dodecyl sulphate-soluble and -insoluble fractions as major end products. Polyacrylamide-gel electrophoresis of sodium dodecyl sulphate-soluble products indicated the formation of mobile components with Mr values between 40 000 and 200 000 (Sepharose CL-6B). The lipid-linked [14C]arabinosyl oligosaccharide possessed properties comparable with those of unsaturated polyisoprenyl pyrophosphoryl derivatives. It was hydrolysed by dilute acid to a charged product (apparent Mr 2300) that could be fractionated in alkali. It was degraded to shorter labelled oligosaccharides by slightly more concentrated acid and eventually to [14C]arabinose as the only labelled component. Susceptibility to acid hydrolysis, and methylation analysis, indicated that the oligosaccharide contained approximately seven sequential alpha-1,5-linked arabinofuranosyl units at the non-reducing end. Several acidic residues appear to be interposed between the terminal arabinosyl units and the charged lipid.     

A protein-glucan intermediate during paramylon synthesis.

A sodium deoxycholate extract containing glucosyltransferase activity was obtained from a particulate preparation from Euglena gracilis. It transferred glucose from UDP-[14C]glucose into material that was precipitated by trichloroacetic acid. This material released beta-(1 leads to 3)-glucan oligosaccharides into solution on incubation with weak acid, weak alkali and beta-(1 leads to 3)-glucosidase. The products of the incubation of the deoxycholate extract with UDP-[14C]glucose were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioactive bands were obtained that had the properties of beta-(1 leads to 3)-glucan covalently linked to protein by a bond labile to weak acid. High-molecular-weight material containing a beta-(1 leads to 3)-glucan was also shown to be present by gel filtration. The bond linking glucan to aglycone is possibly a pyrophosphate linkage. It is proposed that in Euglena gracilis beta-(1 leads to 3)-glucan (paramylon) is synthesized on a protein primer.     

Biosynthesis in vitro of GlcA beta 1-3nLcOse4Cer by a novel glucuronyltransferase (GlcAT-1) from embryonic chicken brain

A novel glucuronyltransferase (GlcAT-1) has been detected in embryonic chicken brains. This enzyme catalyzes the biosynthesis in vitro of glucuronic acid containing glycolipids starting from neolactotetraosylceramide (nLcOse4Cer) and neolactohexaosylceramide (nLcOse6Cer). The activity is present primarily in the Golgi-rich membrane fraction and can be extracted (60%) from the membrane using a neutral detergent, Nonidet P-40, at pH 7.0. The detergent-solubilized GlcAT-1 is stable (70%) at -20 degrees C for at least 4 months. Both membrane-bound GlcAT-1 and solubilized GlcAT-1 show similar pH optima, 6.5-7.0, in HEPES buffer. The Km values were 15 and 200 microM with UDP-[14C] GlcA and nLcOse4Cer, respectively, when the detergent-solubilized supernatant fraction was used as enzyme source. The purified 14C radioactive product that comigrated with chemically characterized GlcA beta 1-3nLcOse4Cer (GlcA-nLc4) also yielded a positive immunostain with monoclonal antibody (human IgM-RI). The anomeric linkage was established as beta-linked GlcA to the terminal galactose of the substrate, as evidenced by 90-99% cleavage of the terminal [14C] GlcA by purified Helix pomatia and limpet glucuronidases. Permethylation studies of the radioactive product obtained from [6-3H]Gal beta 1-4LcOse3Cer and non-radioactive UDP-GlcA showed the presence of 2,4,6-tri-O-methylgalactose in the hydrolyzed enzymatic product. These studies established the structure of the biosynthesized product from nLcOse4Cer as GlcA beta 1-3Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-4Glc-ceramide.     

Incorporation of galactose into galactosyltransferase

Bovine skim milk galactosyltransferase (EC 2.4.1.22) retained its catalytic activity after partial enzymatic removal of sialic acid and galactose. Desialylated and degalactosylated galactosyltransferase was a galactosyl acceptor in the galactosyltransferase reaction. [14C]Galactose from UDP-[14C]galactose was incorporated into the carbohydrate-depleted galactosyltransferase by native galactosyltransferase. The results suggest that galactosyltransferase participates in the biosynthesis of its glycopeptides of the sialic acid-galactose-N-acetylglucosamine type.     

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).     

In vitro biosynthesis of homogalacturonan by a membrane-bound galacturonosyltransferase from epicotyls of azuki bean

A membrane preparation of 7-d-old seedlings from azuki bean (Vigna angularis) contained galacturonosyltransferase (GalAT) capable of transferring galacturonic acid (GalA) from UDP-GalA into polygalacturonic acid (PGA) as an exogenous acceptor. The enzyme was maximally active at pH 6.8-7.8 and 25-35 degrees C in the presence of 5 mM Mn2+ and 0.5% (w/v) Triton X-100. Acid-soluble low-Mr (average Mr 10,000) PGA was a more efficient acceptor substrate than acid-insoluble polymer (Mr 70,000). The apparent Michaelis constants for UDP-GalA and low-Mr PGA were 0.14 mM and 0.02 mg/ml, respectively. Various pectins with different degrees of methyl-esterification (DE) were poor acceptors, and the enzyme activity tended to decrease with decreasing DE of the pectins. The transfer products from incubation of the enzyme with UDP-14C-GalA and the low-Mr PGA yielded 14C-GalA2 as the major product upon digestion with an endopolygalacturonase (EPGase), confirming the incorporation of GalA into PGA through contiguous alpha-1,4-linkages.     

Stimulation of lipid-linked oligosaccharide assembly during oviduct differentiation

Regulation of dolichyl phosphate-linked oligosaccharide assembly has been studied during the course of diethylstilbestrol-induced chick oviduct differentiation. Oviduct membranes from treated chicks form 4.6 times as much GlcNAc-P-P-Dol and GlcNAc2-P-P-Dol upon incubation with UDP-[14C]GlcNAc and MgCl2 than do membranes from untreated chicks. Assembly of oligosaccharide-lipid was studied by incubation of membranes with purified exogenous [14C]GlcNAc2-P-P-Dol and GDP-Man. Man transfer required a divalent cation (10 mM Mg2+) and detergent (0.5% Nonidet P-40 is optimal) and occurs in the presence of amphomycin (500 micrograms/ml). The apparent Km for GDP-Man is 1 microM and for [14C]GlcNAc2-P-P-Dol is 0.45 microM. The products are a series of sequentially formed dolichyl pyrophosphate-linked saccharides up to Man5GlcNAc2, the first of which is Man beta 1,4GlcNAc2. The same products are formed either in the presence or absence of amphomycin. Conversion of GlcNAc2-P-P-Dol to higher oligosaccharides is stimulated 3-fold by estrogen treatment of chicks. Similarly, the conversion of partially purified exogenously added Man beta-[14C]GlcNAc2-P-P-Dol is 4.6-fold higher after diethylstilbestrol treatment.     

Biosynthesis and cell-wall deposition of a pectin–xyloglucan complex in pea

Golgi-enriched enzyme preparations prepared from etiolated pea epicotyls incorporated [U–¹⁴C]galactose from UDP-[U–¹⁴C]galactose into the 1,4--galactan sidechains of a pectin–xyloglucan complex. This complex could bind to paper and was degraded both by pectin-degrading enzymes and by a xyloglucan-specific endoglucanase. Gel permeation chromatography was used to assess the molecular size of the complex and of enzymically-degraded, galactan-containing fragments of it. Etiolated pea stems were labelled with [U–¹⁴C]sucrose for 1 h, and the newly-synthesised cell wall polysaccharides were extracted with EDTA or NaOH and fractionated by ion-exchange chromatography. The NaOH-extracted, acidic radioactive polysaccharides obtained in this way were also degraded both by pectin-degrading enzymes and by xyloglucan-specific endoglucanase. Analysis of the radioactive sugar composition indicated that neutral sugars characteristic of both pectin and xyloglucan were present. Analysis of the total non-radioactive, neutral sugar composition of the NaOH-extracted, acidic cell-wall polysaccharides indicated that pectin–xyloglucan complexes were a general feature of the cell wall in this tissue     

Glycosylation in vitro of Semliki-Forest-virus and influenza-virus glycoproteins and its suppression by nucleotide-2-deoxy-hexose

Cell-free enzyme preparations from cultured fibroblasts infected with Semliki forest virus or fowl plague virus (an influenza A virus) incorporate [14C]-mannose into dolichol-phosphate-mannose, lipid-linked oligosaccharides and into endogenous virus-specific glycoproteins. When GDP-2-deoxy-D-[14C]glucose serves as substrate 2-deoxy-D-[14C]glucose is transferred to dolichol phosphate yielding dolichol-monophosphate-2-deoxy-D-[14C]glucose. UDP-2-deoxy-D-[14C]glucose gives rise also to a lipid which, however, is not a polyprenol derivative. The transfer of [14C]mannose to lipid-extractable fractions and glycoproteins in vitro is blocked by GDP-2-deoxy-D-glucose. It can be restored by exogenous dolichol monophosphate only with regard to the formation of dolichol-monophosphate-[14C]mannose-labelled oligosaccharides into glycoproteins. UDP-2-deoxy-D-glucose has no inhibitory effect on transfer reactions of [14C]mannose from GDP-[14C]mannose into various lipid fractions or into glycoprotein. It is concluded therefore, that the inhibition of glycosylation brought about by 2-deoxyglucose in vivo is caused by an interference of its GDP derivative with the formation of a correct lipid-oligosaccharide.     

Conversion of labeled substrates to sugars, cell wall polysaccharides, and tartaric Acid in grape berries

[U-¹⁴C]Sucrose, myo-[U-¹⁴C]inositol, [6-¹⁴C]- and [U-¹⁴C]glucuronate, UDP-[U-¹⁴C]glucuronate, [U-¹⁴C]gluconate, and l-[1-¹⁴C]ascorbic acid were fed into grape berries, Vitis labrusca L. cv. Delaware, at intervals throughout the ripening process and incorporation of ¹⁴C into several metabolites was studied.     

Glycoproten biosynthesis in Trypanosoma brucei. The glycosylation of Glycoproteins located in and attached to the plasma membrane

1. Glycosyltransferase activity incorporating N-[14C]acetylglucosamine ([14C]GlcNAc) from uridine diphosphate N-[14C]acetylglucosamine (UDP-[14C]GlcNAc) into endogenous proitein acceptors was localized primarily in the plasma membrane of Trypanosoma brucei. 2. The acceptor site for the nucleotide sugar was further localized exclusively to the cytoplasmic face of the plasma membrane. 3. The glycosyltransferase produced elongation of the growing oligosaccharide chains while they were attached to their peptide acceptors. 4. This glycosyltransferase activity was incapable of initiating sugar attachment directly to amino acid residues within peptide acceptors. 5. The dolichyl-phosphate-sugar pathway for glycoprotein biosynthesis was either absent of only present at a very low level in T. brucei when compared to rat liver. 6. All oligosaccharide chains accepting GlcNAc were of the same or very similar lengths. 7. Both O-glycosidic (26%) and N-glycosidic (74%) linkages (exclusive of hydroxylysine attachment) were found. 8. Glycosyltransferase activity required either Mn2+ or Mg2+, had a pH optimum of 6.5 and was temperature-dependent. 9. The kinetics of incorporation were complex, probably a result of multiple acceptors or glycosyltransferases whose activities were characterized by a Km of 30 microM for UDP-GlcNAc with a V of 40 pmol x mg protein -1 x min-1 for the highest affinity system and a Km of approximately 2 mM for UDP-GlcNAc with a V of approximately 400 pmol x mg protein-1 x min-1 for the lowest affinity system. 10. Glycosyltransferases using UDP-GlcNAc, uridine diphosphate glucose, uridine diphosphate galactose and guanidine diphosphate mannose as glycosyl donors were observed. Each peptide acceptor was specific for a singloe labelled sugar in the absence of other unlabelled nucleotide sugars. 11. The final extent of incorporation of GlcNAc was due primarily to exhaustion of peptide acceptor. 12. An inhibitor of UDP-[14C]GlcNAc incorporation into plasma membranes was found in the cytoplasmic fraction.