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1.
1. The principle of radioisotope dilution was applied on a semi-micro scale to the determination of glucosamine and galactosamine in some glycoproteins, such as immunoglobulins, a urinary glycoprotein and blood-group-specific substances. 2. The glycoprotein was hydrolysed in the presence of [1-(14)C]glucosamine or [1-(14)C]galactosamine or both. The amino sugars were made to react with naphthyl isothiocyanate and the products formed were isolated by the method of Scott (1962). The specific radioactivities determined from liquid-scintillation counting and the extinction at 240mmu or 222mmu were used to calculate the content of amino sugars in the protein analysed. 3. Where the values could be compared with those found by other workers, differences were in general not very great. The advantages of the method are that high concentrations of acid can be employed and undesirable side reactions, which may occur with the free sugars, do not affect the results. A potential source of error of the method is discussed.  相似文献   

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Glycation of bovine serum albumin was measured for mannose and fucose at 37 degrees C. Mannose as well as fucose demonstrated an initial rapid increase in rate of formation of total adducts followed by a slower secondary reaction. The equilibrium constant for Schiff base formation was almost two times larger for mannose than fucose, although the Schiff base formed by fucose rearranged 1.5 times faster than that for mannose. Both sugars showed parallel lines for the formation of total and acid stable products after three hours. Discussion integrates new mechanistic data with previously suggested mechanisms.  相似文献   

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Dorsal root ganglia were removed from adult bullfrogs and incubated with [3H]fucose for intervals from 15 min to 1 h, followed by fixation. Some ganglia were post-incubated in the absence of [3H]fucose for up to 17 h. In additional in vivo experiments, young frogs were injected with [3H]fucose, and killed 30 min or 1 h later, and then ganglia were removed and fixed. Electron microscope radioautographs of the ganglia revealed an intense radioautographic reaction over the nuclei of Schwann and satellite cells as early as 5 min after initial exposure to [3H]fucose. At time intervals up to 2 h after initial exposure to [3H]fucose, the silver grains were evenly distributed over both the periphery and internal regions of the nucleus, while at 18 h they were localized to the cell periphery. In occasional cells, the perinuclear space was expanded in some areas and was the site of reaction. In young rats, injected with [3H]galactose and killed 15 min to 5 h later, electron microscope radioautographs revealed heavy reaction over the nuclei of duodenal villous and crypt columnar cells, in which the grains were evenly distributed over both the peripheral and internal regions. In mitotic cells, grains appeared to be associated with the condensed chromatin of forming chromosomes. These results provide strong evidence that glycoproteins exist in the nuclei of the above cell types and that they are actively renewed. The rapid appearance of nuclear reaction after initial exposure to [3H]fucose or [3H]galactose indicates that either these sugars are added to glycoproteins within the nucleus itself or that they migrate rapidly to this site after having been glycosylated elsewhere.  相似文献   

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Patterns of oxidation of dilute solutions of desialylated fetuin and submaxillary mucin by galactose oxidase have been examined. A significant portion (20-40%) of the terminal galactosyls exposed on the glycoproteins, which theoretically were expected to be accessible to the enzyme, was not oxidized. In comparison, galactosyls in oligosaccharides released from completely desialylated glycoproteins were oxidized more effectively with an apparently lower degree of crypticity to the enzyme. Partial desialylation usually resulted in a reduction of both the rate and the final level of substrate oxidation. A second cycle of oxidation of a desialylated substrate earlier oxidized by galactose oxidase and then reduced by NaB3H4 revealed a selectivity in the pattern of galactosyl oxidation. The same galactosyl residues oxidized in the first cycle were again the most susceptible to oxidation in the second cycle, leaving unmodified the same fraction of galactosyls throughout both cycles. The relevance of these results to the application of the galactose oxidase-NaBH4 procedure for detecting and measuring desialylated glycoconjugates in solution and in biological membranes is discussed.  相似文献   

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O-Linked fucose in glycoproteins from Chinese hamster ovary cells   总被引:1,自引:1,他引:1  
We report our discovery that many glycoproteins synthesizedby Chinese hamster ovary (CHO) cells contain fucose in O-glycosidiclinkage to polypeptide. To enrich for the possible presenceof O-linked fucose, we studied the lectin-resistant mutant ofCHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferaseI and are therefore unable to synthesize complex-type N-linkedoligosaccharides. Lec1 cells were metabolically radiolabelledwith [6-3H]fucose and total glycoproteins were isolated. Glycopeptideswere prepared by proteolysis and fractionated by chromatographyon a column of concanavalin A (Con A)— Sepharose. Thesets of fractionated glycopeptides were treated with mild base/borohydrideto effect the ß-elimination reaction and release potentialO-linked fucosyl residues. The ß-elimination produced[3H]fucitol quantitatively from [3H]fucose-labelled glycopeptidesnot bound by Con A-Sepharose, whereas none was generated bytreatment of glycopeptides bound by the lectin. The total [3H]fucose-labelledglycoproteins from Lec1 cells were separated by SDS—PAGEand detected by fluorography. Treatment of selected bands ofdetectable glycoproteins with mild base/borohydride quantitativelygenerated [3H]fucitol. Pretreatment of the glycoproteins withN-glycanase prior to the SDS—PAGE method of analysis causedan enrichment in the percentage of radioactivity recovered as[3H]fucitol. Trypsin treatment of [3H]fucose-labelled intactCHO cells released glycopeptides that contained O-linked fucose,indicating that it is present in surface glycoproteins. Thesefindings demonstrate that many glycoproteins from CHO cellscontain O-linked fucosyl residues and raise new questions aboutits biosynthesis and possible function. fucose glycoproteins monosaccharide O-linked  相似文献   

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We developed a convenient and specific method for the determination of mucin-type glycoproteins using galactose oxidase and horseradish peroxidase on the basis of the contents of galactosyl and N-acetylgalactosaminyl residues in glycoproteins. Galactose and galactosamine residues released from glycoproteins after hydrolysis were oxidized with galactose oxidase and subsequently the resultant hydrogen peroxide was determined by a combination of horseradish peroxidase and 3-(p-hydroxyphenyl) propionic acid as a fluorogenic substrate. The contents of galactose/galactosamine residues in N- and O-glycans, as determined by the galactose oxidase-peroxidase method, were in good agreement with those described in the previous reports. We applied the present method to determine mucin-type glycoproteins secreted from rat gastric mucosa by stimulation with misoprostol, a prostaglandin E(1) analogue in vivo. Thus, the galactose oxidase-peroxidase method is useful for the determination of mucin-type glycoproteins in biological materials.  相似文献   

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The purple pigmented bacterium Chromobacterium violaceum is a dominant component of tropical soil microbiota that can cause rare but fatal septicaemia in humans. Its sequenced genome provides insight into the abundant potential of this organism for biotechnological and pharmaceutical applications and allowed an ORF encoding a protein that is 60% identical to the fucose binding lectin (PA-IIL) from Pseudomonas aeruginosa and the mannose binding lectin (RS-IIL) from Ralstonia solanacearum to be identified. The lectin, CV-IIL, has recently been purified from C. violaceum [Zinger-Yosovich, K., Sudakevitz, D., Imberty, A., Garber, N. C., and Gilboa-Garber, N. (2006) Microbiology 152, 457-463] and has been confirmed to be a tetramer with subunit size of 11.86 kDa and a binding preference for fucose. We describe here the cloning of CV-IIL and its expression as a recombinant protein. A complete structure-function characterization has been made in an effort to analyze the specificity and affinity of CV-IIL for fucose and mannose. Crystal structures of CV-IIL complexes with monosaccharides have yielded the molecular basis of the specificity. Each monomer contains two close calcium cations that mediate the binding of the monosaccharides, which occurs in different orientations for fucose and mannose. The thermodynamics of binding has been analyzed by titration microcalorimetry, giving dissociation constants of 1.7 and 19 microM for alpha-methyl fucoside and alpha-methyl mannoside, respectively. Further analysis demonstrated a strongly favorable entropy term that is unusual in carbohydrate binding. A comparison with both PA-IIL and RS-IIL, which have binding preferences for fucose and mannose, respectively, yielded insights into the monosaccharide specificity of this important class of soluble bacterial lectins.  相似文献   

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A precise and accurate gas—liquid chromatographic (GLC) method has been developed for the quantitative analysis of the neutral sugars -fucose (6-deoxygalactose), mannose, galactose, and glucose in ethanol precipitates of human serum proteins. The chromatographic conditions and sample preparation resulted in short analysis times (20 min per run) and made routine analyses practicable (twelve samples per day). The alditol acetate derivatization yielded single derivatives for each sugar. Complete separation was achieved on a 2.0 m × 2 mm I.D. column with 2.0% Silar-7 CP on Chromosorb W AW 80–100 mesh. The results of hydrolysis showed that the release of fucose and galactose preceded the release of mannose. Hydrolysis with AG 50W-X8 (H+) ion-exchange resin in 0.5 N HCI at 100° for 7 h optimized glycosidic bond cleavage with only minimal destruction of fucose, mannose and galactose. A combination of strong cation- and anion-exchange resin columns was used to remove chromatographic background of peptides, amino acids, amino sugars, and inorganic ions. An average R.S.D. of less than 4% with recovery of <86% for the three sugars was achieved. The homogeneity of the chromatographic peaks for the neutral sugars of normal human serum glycoproteins was confirmed by GLC—mass spectrometry. Significantly elevated ratios of fucose, galactose, and mannose to serum protein were observed for patients with small cell lung and ovarian carcinomas.  相似文献   

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The molecular and crystal structures of 12 N-aryl-beta-D-glycopyranosylamines have been determined by X-ray crystallography. Six of these are mannose derivatives, the N-p-bromophenyl (1), N-p-tolyl (2), N-m-chlorophenyl (3), N-p-methoxyphenyl (4), N-o-chlorophenyl (5), and N-o-tolyl (6) derivatives that are formed by reaction with the corresponding substituted anilines. The remaining six are galactose derivatives, the N-phenyl (7), N-p-chlorophenyl (8), N-p-bromophenyl (9), N-p-iodophenyl (10), N-p-nitrophenyl (11) and N-p-tolyl (12), derivatives prepared similarly. Compounds 1-3 assume the same packing arrangement. Compounds 4, 5, and 6 assume unique packing arrangements, although that assumed by 4 is closely related to that assumed by 1-3. Compounds 7-11 assume the same packing arrangement; that assumed by 12 is closely related to that assumed by 7-11. That the same packing arrangements can be maintained in spite of substantial changes in the electronic and steric nature of the substituent on the aryl ring reflects the strength of the hydrogen bond network connecting the monosaccharide portions of the molecules in the solid state. A hydrogen bonding motif found in all six mannose structures is a mutual interaction between translationally related molecules involving O-3-H...O-5 and O-6-H...O-4 hydrogen bonds. The recurrence of this motif throughout this group of mannosylamines suggests that it is an especially favorable interaction that might be expected to occur also in related macromolecular systems.  相似文献   

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The parameters that affect the interaction of ligands with a fucose-binding lectin from rat liver have been examined. 125I-Fucosyl-bovine serum albumin (Fuc-BSA) containing 50 residues of fucose/molecule was used as the standard ligand. At low initial concentrations of ligand (10 ng/ml) and lectin (140 ng/ml), the reaction reaches equilibrium at pH 7.8, 23 degrees C, within 40 min. The binding of ligands is Ca2+ dependent with half-maximal binding occurring at 54 microM Ca2+; of several metal ions tested, only Sr2+ partially replaced Ca2+. Binding was maximal between pH 7.6 and 8.6, fell slightly up to pH 10, but fell markedly below pH 7. The lectin-ligand complexes dissociated at low pH, on removal of Ca2+, or in the presence of a large excess of competing ligand. The apparent association constant (Ka) for Fuc-BSA was 1.75 X 10(8) M-1. The fucose content of the Fuc-BSA also influenced binding, with little apparent binding below 24 fucose residues/molecule and maximal binding from 40 to 50 fucose residues/molecule. With knowledge of the parameters influencing binding, sensitive reproducible assays for the lectin were developed. The binding specificity of the lectin was examined by measuring the inhibition of 125I-Fuc-BSA binding by neoglycoproteins, monosaccharides, and glycosides or by direct binding of neoglycoproteins. Galactosides and beta-linked fucosides were the best ligands among the neoglycoproteins, with much weaker binding by mannosyl- or N-acetylglucosaminyl-BSA. On the basis of the pattern of inhibition of Fuc-BSA binding by various monosaccharides and glycosides, it is possible to propose the conformations of saccharides that best fit the lectin-binding site. The C1 conformation of N-acetyl-D-galactosamine fits best, although other not obviously related monosaccharides such as L-fucose, L-arabinose, and D-mannose can also assume conformations that permit them to be effective inhibitors. The pattern of binding of neoglycoproteins to the lectin differs from that of other pure hepatic lectins. Thus, the fucose lectin has a high affinity for Fuc-BSA and galactosyl-BSA but a low affinity for N-acetylglucosaminyl-BSA. The galactose lectin binds only galactosyl-BSA and shows little binding with either N-acetylglucosaminyl-BSA or Fuc-BSA. In contrast, the mannose/N-acetylglucosamine lectin binds N-acetylglucosaminyl-BSA and Fuc-BSA but not galactosyl-BSA.  相似文献   

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Peptide N-glycosidase from Flavobacterium meningosepticum cleaves complex as well as neutral glycoproteins (Plummer, T.H., Jr., Elder, J.H., Alexander, S., Phelan, A.W., and Tarentino, A.L. (1984) J. Biol. Chem. 259, 10700-10704). Examples of neutral glycoprotein substrates include ribonuclease B (one high mannose oligosaccharide chain) and yeast external invertase (nine chains/invertase subunit). The rate of deglycosylation by the glycosidase was greatly enhanced if the glycoprotein substrate was denatured prior to enzyme treatment, from a low of 11-fold for external invertase to a high of 844-fold for ribonuclease B. Peptide N-glycosidase F was unable to cleave the asparaginyl-N-acetylglucosamine bond in endo-beta-N-acetylglucosaminidase H-modified external invertase or ribonuclease B, although that in similarly modified glycopeptide substrate was cleaved. Ribonuclease B was digested sequentially with various exoglycosidases to produce an oligosaccharide chain of varied length. Using the resulting forms of ribonuclease B as substrates for peptide N-glycosidase F, the minimum oligosaccharide chain for cleavage was the di-N-acetyl-chitobiosyl core unit.  相似文献   

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