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1.
Biosynthesis of the fucose-containing xyloglucan nonasaccharide by pea microsomal membranes 总被引:4,自引:3,他引:1
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Pea microsomal membranes catalyze the transfer of [14C]fucose (Fuc) from GDP-[U-14C]fucose, with or without added unlabeled UDP-glucose (Glc), UDP-xylose (Xyl) or UDP-galactose (Gal), to an insoluble product with properties characteristic of xyloglucan. After digestion of the ethanol-insoluble pellet with Streptomyces griseus endocellulase, [14C] fucose residues occur exclusively in a fragment corresponding in size to the xyloglucan nonasaccharide, Glc4 Xyl3 Gal Fuc. This fragment contains a single labeled fucose residue per oligomer, α-linked in a terminal nonreducing position. By comparison, in incubations where GDP-[14C] fucose is absent and replaced by UDP-[3H]xylose, the maximum size of labeled oligosaccharide found following cellulase digestion of products is an octasaccharide. In the presence of both GDP-[14C]fucose and UDP-[3H]xylose, a nonasaccharide containing the two labels is produced. Fucose and xylose residues are transferred within a few minutes to acceptor molecules of molecular weight up to 300,000. Such products do not elongate detectably over 60 minutes of incubation. The data support the conclusion that the nonasaccharide subunit of xyloglucan may be generated in vitro by transfucosylation to preformed acceptor chains, and that its synthesis is dependent on the inclusion of exogenous GDP-fucose. 相似文献
2.
The transfer of [14C]fucose from GDP-[U-14C]fucose to endogenous and exogenous acceptors by particulate and solubilized preparations from mouse brain is described. Suspensions of brain microsomes incorporated [14C]fucose into a heterogenous group of glycoprotein products, which have a distribution on gel electrophoresis similar to those synthesized in vivo. Fucosyl transferase, extracted from brain microsomes by Triton X-100, transferred [14C]fucose from GDP-[U-14C]fucose to terminal galactose residues exposed by mild acid hydrolysis of porcine plasma glycoprotein. Comparison of the specific activities of the solubilized fucosyl transferase from a number of organs showed that, in the presence of the exogenous acceptor which was used, the transferase of brain was more active than the transferases from all other organs tested, with the exception of kidney. Examination of subcellular fractions of brain, with endogenous and exogenous acceptors, showed that activity was limited to fractions containing microsomal membranes, whereas synaptosomal and other fractions were virtually inactive. 相似文献
3.
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. 相似文献
4.
A macromolecular complex composed of xyloglucan and cellulose was obtained from elongating regions of etiolated pea (Pisum sativum L. var. Alaska) stems. Xyloglucan could be solubilized by extraction of this complex with 24% KOH-0.1% NaBH4 or by extended treatment with endo-1,4-β-glucanase. The polysaccharide was homogeneous by ultracentrifugal analysis and gel filtration on Sepharose CL-6B, molecular weight 330,000. The structure of pea xyloglucan was examined by fragmentation analysis of enzymic hydrolysates, methylation analysis, and precipitation tests with fucose- or galactose-binding lectins. The polysaccharide was composed of equal amounts of two subunits, a nonasaccharide (glucose/xylose/galactose/fucose, 4:3:1:1) and a heptasaccharide (glucose/xylose, 4:3), which appeared to be distributed at random, but primarily in alternating sequence. The xyloglucan:cellulose complex was examined by light microscopy using iodine staining, by radioautography after labeling with [3H]fucose, by fluorescence microscopy using a fluorescein-lectin (fucose-binding) as probe, and by electron microscopy after shadowing. The techniques all demonstrated that the macromolecule was present in files of cell shapes, referred to here as cell-wall `ghosts,' in which xyloglucan was localized both on and between the cellulose microfibrils. Since the average chain length of pea xyloglucan was many times the diameter of cellulose microfibrils, it could introduce cross-links by binding to adjacent fibrils and thereby contribute rigidity to the wall. 相似文献
5.
Synthesis of glycoproteins in a single identified neuron of Aplysia californica 总被引:6,自引:1,他引:5
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Incorporation of L-[3H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [3H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [3H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [3H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [3H]fucose, gel profiles of particulate fractions labeled with [14C]valine were much more complex. 相似文献
6.
Mature seeds of Tropaeolum majus L. contain the cell wall polysaccharide xyloglucan (amyloid), protein and lipid as storage substances. The transitory occurrence of starch during the process of seed development could be substantiated.[U-14C]-labelled xylose, glucose and glucuronic acid were fed to ripening seeds and the incorporation of radioactivity into xyloglucan, starch and the sugar nucleotide fraction of the cotyledons was determined. The results indicate that exogenous supplied xylose is not incorporated directly into xyloglucan, but is transformed to glucose before incorporation into xyloglucan and starch. Radioactivity from glucuronic acid was predominantly found in the xylose moiety of xyloglucan. Incubation of seeds with [6-14C]-labelled glucose resulted in an incorporation of labelled hexoses into amyloid and starch, whereas xylose residues of amyloid remained unlabelled.Abbreviations p.a.
post anthesis
- UDP
uridine 5-diphosphate
- GDP
guanosine 5-diphosphate
- TLC
thin layer chromatography
- HPLC
high pressure liquid chromatography 相似文献
7.
《Phytochemistry》2012
Angiosperms possess a retaining trans-α-xylosidase activity that catalyses the inter-molecular transfer of xylose residues between xyloglucan structures. To identify the linkage of the newly transferred α-xylose residue, we used [Xyl-3H]XXXG (xyloglucan heptasaccharide) as donor substrate and reductively-aminated xyloglucan oligosaccharides (XGO–NH2) as acceptor. Asparagus officinalis enzyme extracts generated cationic radioactive products ([3H]Xyl·XGO–NH2) that were Driselase-digestible to a neutral trisaccharide containing an α-[3H]xylose residue. After borohydride reduction, the trimer exhibited high molybdate-affinity, indicating xylobiosyl-(1→6)-glucitol rather than a di-xylosylated glucitol. Thus the trans-α-xylosidase had grafted an additional α-[3H]xylose residue onto the xylose of an isoprimeverose unit. The trisaccharide was rapidly acetolysed to an α-[3H]xylobiose, confirming the presence of an acetolysis-labile (1→6)-bond. The α-[3H]xylobiitol formed by reduction of this α-[3H]xylobiose had low molybdate-affinity, indicating a (1→2) or (1→4) linkage. In NaOH, the α-[3H]xylobiose underwent alkaline peeling at the moderate rate characteristic of a (1→4)-disaccharide. Finally, we synthesised eight non-radioactive xylobioses [α and β; (1↔1), (1→2), (1→3) and (1→4)] and found that the [3H]xylobiose co-chromatographed only with (1→4)-α-xylobiose. We conclude that Asparagus trans-α-xylosidase activity generates a novel xyloglucan building block, α-d-Xylp-(1→4)-α-d-Xylp-(1→6)-d-Glc (abbreviation: ‘V’). Modifying xyloglucan structures in this way may alter oligosaccharin activities, or change their suitability as acceptor substrates for xyloglucan endotransglucosylase (XET) activity. 相似文献
8.
Soybean (Glycine max) membranes co-equilibrating with Golgi vesicles in linear sucrose gradients contained UDP-glucuronate carboxy-lyase and xyloglucan synthase activities. Digitonin solubilized and increased the activity of the membrane-bound UDP-glucuronate carboxy-lyase. UDP-xylose did not inhibit the transport of UDP-glucuronate into the lumen of Golgi vesicles but repressed the decarboxylation of the translocated UDP-glucuronate. The results suggest that UDP-glucuronate is transported into the vesicles by a specific carrier and decarboxylated to UDP-xylose within the lumen. On incubation of UDP-[14C]glucuronate with Golgi membranes in the presence of UDP-glucose, [14C]xylose-labeled xyloglucan was formed. Although the Km value of UDP-glucuronate for the decarboxylation was 240 micromolar, the affinity of UDP-glucuronate for xyloglucan formation (31 micromolar) was similar to that of UDP-xylose (28 micromolar), suggesting a high turnover of UDP-xylose. The biosynthesis of UDP-xylose from UDP-glucuronate probably occurs in Golgi membranes, where xyloglucan subsequently forms from UDP-xylose and UDP-glucose. 相似文献
9.
Distribution of secretory component in hepatocytes and its mode of transfer into bile 总被引:4,自引:1,他引:3
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Barbara M. Mullock Richard H. Hinton Miloslav Dobrota Jane Peppard Eva Orlans 《The Biochemical journal》1980,190(3):819-826
Immunoglobin A in bile and other external secretions is mostly bound to a glycoprotein known as secretory component. This glycoprotein is not synthesized by the same cells as immunoglobulin A and is not found in blood. We now report the mechanism by which secretory component reaches the bile and describe its function in immunoglobulin A transport across the hepatocyte. Fractionation of rat liver homogenates by zonal centrifugation was followed by measurement of the amounts of secretory component in the various fractions by rocket immunoelectrophoresis. Secretory component was found in two fractions. One of these was identified as containing Golgi vesicles from its isopycnic density and appearance in the electron microscope; the other contained principally fragments of the plasma membrane of the sinusoidal face of the hepatocyte, as shown by its particle size and content of marker enzymes. Only the latter fraction bound 125I-labelled immunoglobulin A added in vitro. At 5min after intravenous injection of [14C]fucose, the secretory component in the Golgi fraction was labelled, but not that in the plasma membrane. The secretory component in the sinusoidal plasma membrane did, however, become labelled before the first labelled secretory component appeared in bile, about 30min after injection. We suggest that fucose is added to the newly synthesized secretory component in the Golgi apparatus. The secretory component then passes, with the other newly secreted glycoproteins, to the sinusoidal plasma membrane. There it remains bound but exposed to the blood and able to bind any polymeric immunoglobulin A present in serum. The secretory component then moves across the hepatocyte to the bile-canalicular face in association with the endocytic-shuttle vesicles which carry immunoglobulin A. Hence there is a lag before newly synthesized secretory component appears in bile. 相似文献
10.
Patrick Campbell Ingvar Jacobsson Laure Benzing-Purdie Lennart Rodén John H. Fessler 《Analytical biochemistry》1984,137(2):505-516
The formation of most connective tissue polysaccharides is initiated by transfer of d-xylose from UDP-d-xylose to specific serine residues in the core proteins of the putative proteoglycans. The substrate specificity of the xylosyltransferase catalyzing this reaction has not yet been examined in detail, but it appears that a -Ser-Gly- pair is an essential part of the substrate structure. Since the preparation of the known acceptors (e.g., Smith-degraded or HF-treated cartilage proteoglycan) involves a substantial effort, we have searched for readily available proteins with the -Ser-Gly-sequence, which might serve as alternative substrates. In the present work, it was found that silk fibroin from Bombyx mori, which consists, in large part, of the repeating hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly, is an excellent substrate for the xylosyltransferase from embryonic chick cartilage. Pieces of silk were used directly in the reaction mixtures, and [14C]xylose transferred from UDP-d-[14C]xylose was measured by liquid scintillation spectrometry after rinsing the silk in 1 m NaCl and water. Substantially greater incorporation was observed with preparations of silk or fibroin which had been dissolved in 60% LiSCN and subsequently dialyzed exhaustively or diluted appropriately. Under standard reaction conditions, the Vmax for fibroin was 531 pmol/h/mg enzyme protein, as compared to 223 pmol/h/mg for Smith-degraded proteoglycan. Km values were 182 mg/liter (fibroin) and 143 mg/liter (Smith-degraded proteoglycan). The product of [14C]xylose transfer to silk was alkali labile, and [14C]xylitol was formed when [14C]xylosylsilk was treated with borohydride in alkali. Proteolytic digestion with papain, Pronase, leucine aminopeptidase, and carboxypeptidase A yielded a radioactive product which was identified as [14C]xylosylserine by electrophoresis and chromatography. The identity of the isolated [14C]xylosylserine was further supported by its resistance to treatment with alkali (0.5 m KOH: 100°C; 8h) and by acid hydrolysis which yielded [14C]xylose. Tryptic and chymotryptic fragments from fibroin were also good xylose acceptors and had Vmax values 60–70% of that observed for the intact protein. Substantial acceptor activity was displayed also by the sericin fraction of silk and by the silk sequence hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly; the latter had a Vmax value close to 20% of that of intact fibroin. 相似文献
11.
A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-14C]mevalonic acid, [1-14C]acetate, [3-14C]pyruvate, [U-14C]sucrose, [U-14C]glucose, [U-14C]xylose, [U-14C]glyoxylate, [2,3-14C]succinic acid, [1-14C]glycerol [U-14C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-14C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-14C]sucrose, [U-14C]glucose, [U-14C]xylose [1-14C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of 14C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed. 相似文献
12.
A particulate enzyme preparation capable of catalyzing the transfer of d-[U-14C]apiose and d-[U-14C]xylose from uridine 5′-(α-d-[U-14C]apio-d-furanosyl pyrophosphate) (UDP[U-14C]Api) and uridine 5′-(α-d-[U-14C]xylopyranosyl pyrophosphate) (UDP[U-14C]Xyl) to endogenous acceptor molecules was isolated from Lemna minor. The two enzymes were named UDP-d-apiose:acceptor d-apiosyltransferase and UDP-d-xylose:acceptor d-xylosyltransferase and were associated with particulate material sedimenting between 480 and 34,800g. The rate of d-[U-14C]apiose or d-[U-14C]xylose incorporation was proportional to the quantity of enzyme preparation used and was constant with time to 1.5 min. Both enzymes showed a pH optimum of 5.7 in citrate-phosphate buffer. The d-apiosyltransferase has a Km for UDP[U-14C]Api of 4.9 μm. Bovine serum albumin and sucrose stimulated the rate of incorporation of both pentoses. Both enzymes rapidly lost activity; with our best conditions, approximately 50% of each enzyme activity was lost in 6 min at 25 °C or in 3 h at 4 °C. Incorporation of d-[U-14C]apiose was obtained in the absence of added uridine 5′-(α-d-galactopyranosyluronic acid pyrophosphate) (UDPGalUA); however, the addition of UDPGalUA not only almost doubled the rate of incorporation, but also increased the total incorporation of d-[U-l4C]apiose and extended the proportional range of incorporation at 25 °C from 1.5 to 2 min. 相似文献
13.
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). 相似文献
14.
The transfer of mannose from GDP[14C]mannose to lipid and to insoluble polymer by a particulate preparation of Phaseolus aureus has been investigated. The evidence favours the lipid being a prenol phosphate mannose. Of a range of prenol phosphates tried, betulaprenol phosphate was the most effective exogenous acceptor of mannose. Most of the insoluble [14C]polymer formed was glycoprotein in nature although small quantities of 14C were associated with glucomannan and galactoglucomannan fractions. Time studies failed to reveal a typical precursor-product relationship between the lipid and polymer fractions but on incubation of [14C]mannolipid with the particulate fraction a small transfer (0·5–0·7%) of [14C] to polymer was detected. p-Hydroxymercuribenzoate inhibited (by 90%) the transfer of [14C] from GDP[14C]-mannoseto polymer and simultaneously increased (3-fold) the [14C] recovered in the lipid fraction. The effect was nullified by mercaptoethanol. Attempts to solubilize the transfer system were only partially successful. The formation of a chromatographically identical mannolipid was demonstrated in particulate fractions of Codium fragile and tomato roots. 相似文献
15.
Schistosoma mansoni: glycosyl transferase activity and the carbohydrate composition of the tegument.
Homogenates of adult Schistosoma mansoni contain enzymes which transferred [14C]mannose, [14C]glucose, and [14C]galactose from GDP-[U-14C]mannose, UDP-[U-14C]glucose, and UDP-[U-14C]galactose respectively to a lipid acceptor; in comparison, free [14C]mannose, GDP-[U-14C]fucose, and UDP-[U-14C]acetyl-glucosamine were poorly transferred. The lipid acceptor is believed to be an intermediate in the glycosylation of the worm's glycoproteins and in the biosynthesis of oligosaccharides and glycolipids. The tegument of adult worms was isolated by the freeze-thaw procedure and sugars associated with macromolecules in this fraction were analyzed; the major monosaccharide components were glucose, galactose, and mannose. These results suggest that the mechanism of glycosylation of the adult schistosome's tegumental macromolecules may occur through the glycosyl transferase system. The schistosome mannosyl transferase (EC 2.4.1), which is membrane bound was solubilized with 0.1% Triton X-100 without loss of activity; after density gradient centrifugation there was a peak of enzymic activity in a region of density 1.08, which could not be associated with any particular organelle. 相似文献
16.
When UDP-[14C]glucose or UDP-[14C]xylose was incubated witha particulate fraction from soybean cells, radioactive polymerswere synthesized. On digestion with Aspergillus oryzae enzymes,these polymers gave 14C-monosaccharides and a 14C-disaccharidewith chromatographic and electrophoretic mobilities indistinguishablefrom those of authentic isoprimeverose (6-O--D-xylopyranosyl-D-glucopyranose).The disaccharide consisted of xylose and glucose, and the latterwas located at the reducing end. Evidence that the disaccharideis isoprimeverose was provided by methylation analysis. Hydrolysisof the methylated disaccharide yielded 2,3,4-tri-O-methyl-D-xyloseand 2,3,4-tri-O-methyl-D-glucose. Thus, incorporation of radioactivityinto isoprimeverose, the smallest structural unit of xyloglucan,suggests that xyloglucan is synthesized in vitro from UDP-glucoseand UDP-xylose. (Received November 20, 1980; Accepted February 14, 1981) 相似文献
17.
Glycoprotein metabolism in developing mouse brain 总被引:3,自引:3,他引:0
—Incorporation of [14C]fucose or [14C]glucosamine into the glycoproteins of developing mouse brain was studied using polyacrylamide gel electrophoresis. Between 1 and 10 days after birth two fractions of soluble glycoproteins were extensively labelled, but by 15 days after birth incorporation into these fractions was no longer prominent. These glycoproteins have apparent molecular weights in the range of 150,000-250,000, as estimated by the electrophoretic procedure. The more rapidly migrating fraction has a half-life of about 1 week whereas the other is far more stable. 相似文献
18.
When [glucitol-3H]XXFGol (a NaB3H4-reduced xyloglucan nonasaccharide) was applied to excised shoots of pea (Pisum sativum L. cv. Progress) at the base of the epicotyl, it inhibited growth in the elongation zone, 4–5 cm distal. Experiments were
conducted to discover whether such 3H-oligosaccharides are translocated intact over this distance, or whether an intercellular second messenger would have to
be postulated. After 24 h, 3H from [glucitol-3H]XXFGol and [glucitol-3H]XXXGol showed U-shaped distributions, with most 3H at the base and apex of the stem. Radioactivity from [fucosyl-3H]XXFG and [xylosyl-3H]XXFG also moved acropetally, but did not concentrate at the apex, possibly owing to removal from the transpiration stream
of fucose and xylose formed by partial hydrolysis of XXFG en route. When 10−7 M [glucitol-3H]XXFGol was supplied, about 14 fmol · seedling–1 of apparently intact [3H]XXFGol was extractable from the elongation zone after 24 h. Larger amounts of degradation products were extractable (including
free [3H]glucitol) and some wall-bound 3H-hemicellulose was present (presumably formed by the oligosaccharides acting as acceptor substrates for transglycosylation).
We conclude that biologically active oligosaccharides of xyloglucan can move through the stem acropetally and that they are
maintained at low steady-state concentrations by both hydrolysis and transglycosylation.
Received: 1 April 1997 / Accepted: 28 May 1997 相似文献
19.
Cell‐wall components are hydrolysed by numerous plant glycosidase and glycanase activities. We investigated whether plant enzymes also modify xyloglucan structures by transglycosidase activities. Diverse angiosperm extracts exhibited transglycosidase activities that progressively transferred single sugar residues between xyloglucan heptasaccharide (XXXG or its reduced form, XXXGol) molecules, at 16 μm and above, creating octa‐ to decasaccharides plus smaller products. We measured remarkably high transglycosylation:hydrolysis ratios under optimized conditions. To identify the transferred monosaccharide(s), we devised a dual‐labelling strategy in which a neutral radiolabelled oligosaccharide (donor substrate) reacted with an amino‐labelled non‐radioactive oligosaccharide (acceptor substrate), generating radioactive cationic products. For example, 37 μm [Xyl‐3H]XXXG plus 1 mm XXLG‐NH2 generated 3H‐labelled cations, demonstrating xylosyl transfer, which exceeded xylosyl hydrolysis 1.6‐ to 7.3‐fold, implying the presence of enzymes that favour transglycosylation. The transferred xylose residues remained α‐linked but were relatively resistant to hydrolysis by plant enzymes. Driselase digestion of the products released a trisaccharide (α‐[3H]xylosyl‐isoprimeverose), indicating that a new xyloglucan repeat unit had been formed. In similar assays, [Gal‐3H]XXLG and [Gal‐3H]XLLG (but not [Fuc‐3H]XXFG) yielded radioactive cations. Thus plants exhibit trans‐α‐xylosidase and trans‐β‐galactosidase (but not trans‐α‐fucosidase) activities that graft sugar residues from one xyloglucan oligosaccharide to another. Reconstructing xyloglucan oligosaccharides in this way may alter oligosaccharin activities or increase their longevity in vivo. Trans‐α‐xylosidase activity also transferred xylose residues from xyloglucan oligosaccharides to long‐chain hemicelluloses (xyloglucan, water‐soluble cellulose acetate, mixed‐linkage β‐glucan, glucomannan and arabinoxylan). With xyloglucan as acceptor substrate, such an activity potentially affects the polysaccharide’s suitability as a substrate for xyloglucan endotransglucosylase action and thereby modulates cell expansion. We conclude that certain proteins annotated as glycosidases can function as transglycosidases. 相似文献
20.
Robert P. Metzger Kathleen D. Edwards Cecilia C. Nixon Patrick W. Mobley 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,629(3):482-489
The production of 14CO2 from l-[1-14C]fucose and d-[1-14C]arabinose has been studied in five mammalian species.Cats, guinea pigs, mice, and rabbits respired about 22% of the label of l[1-14C]fucose or of d-[1-14C]arabinose within 6 h after intraperitoneal injection of the sugar. Rats respired only 1.5% of the l-fucose label and 5% of the d-arabinose label in the same time period.Liver homogenates from cat, guinea pig, and rabbit produced significantly more 14CO2 from l-[1-14C]fucose or d-[1-14C]arabinose than mouse or rat liver homogenates. Unlike those of the other species, guinea pig liver homogenates had very low l-fucose dehydrogenase activity.The results suggest that substantial catabolism of l-fucose and d-arabinose occurs in the tissues of some animal species. Investigators wishing to employ l-fucose as a tracer of glycoprotein metabolism must, therefore, ensure that the species that they employ does not metabolize l-fucose to products interfering with their studies. 相似文献