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1.
An acidic heteropolysaccharide preparation derived from the mycelium of Fusarium sp. M7-1 was fractionated into two fractions, precipitable and nonprecipitable, by treatment with cetyltrimethylammonium bromide (Cetavlon). These two fractions were further purified to apparent homogeneity on ultracentrifugation by treatment with charcoal and gel filtration chromatographies. Two glycoproteins, precipitable GP I and nonprecipitable GP II, were obtained. The molecular weights of GP I and GP II were estimated to be about 8.8 x 10(4) and 3.7 x 10(4), respectively, on gel filtration chromatography. Both GP I and GP II contained a high proportion of serine and threonine. Treatment of GP I and GP II with alkaline solution resulted in an increase in absorbance at 240 nm. Alkaline borohydride treatment markedly decreased the number of seryl and threonyl residues and resulted in an increase in alanine and the formation of 2-aminobutyric acid. It also resulted in release of low and high molecular weight carbohydrate chains. From these results, we conclude that both GP I and GP II are glycoproteins with carbohydrate chains attached to the protein moiety through O-glycosidic linkages to the hydroxyl group of serine and/or threonine. 相似文献
2.
A large number of oligosaccharide fractions having blood-group Ii activity were obtained by degradation with alkaline borohydride of sheep gastric-glycoproteins which had been enriched for these blood-group activities by affinity chromatography on an anti-I adsorbent column. The approximate molecular weights of the oligosaccharides were estimated from their elution profiles on Bio-Gel P4 and from monosaccharide compositions. The fractions of smallest molecular weight with both I and i activities were mixtures of hexa- to octa-saccharides. From a comparison of the inhibitory activities of these with fractions of higher molecular weight and with synthetic oligosaccharides, it was concluded that the antigenic determinant recognised by anti-I Ma is a trisaccharide, whereas those recognised by other types of anti-I and by anti-i antibodies are longer than trisaccharides. 相似文献
3.
Horse B-active and non-B-active glycoproteins from gastric mucosae are indistinguishable in their precipitating abilities with concanavalin A, anti-BP1, type XIV horse antipneumococcal serum, the lectin from Lotus tetragonolobus and a group 1 anti-I serum, Ma; no Lea or Leb activity was found. Each was subjected to catalyzed release of its oligosaccharide chains by 0.05 n NaOH in 1 m NaBH4. Destruction of serine, threonine and 2-acetamido-2-deoxygalactopyranose (dGalNAc) was associated with production of alanine, α-aminobutyric acid and N-acetyl-d-galactosaminitol, as expected for a carbohydrate to peptide linkage via dGalNAc to serine or threonine. No evidence of basecatalyzed peeling was seen. Bio-Gel P-2 elution patterns of the salt-free oligosaccharides from the two preparations were compared. Unlike results obtained with human ovarian cyst substances, very little material was excluded. The largest-size chains are in the range of deca- or dodecasaccharides, and a reduced octasaccharide was isolated. The four most abundant amino acids in both B-active and non-B-active materials are threonine, serine, proline and glutamic acid, which together account for 60% of the weight of amino acids. 相似文献
4.
F Downs C Peterson V L Murty W Pigman 《International journal of peptide and protein research》1977,10(4):315-322
The carbohydrate side chains of mucus-type glycoproteins are O-glycosidic bonds between N-acetylgalactosamine to the hydroxyl groups of serine and threonine in the protein core. The alkaline catalyzed beta-elimination reaction, in the presence of sodium borohydride, is used for determining the number of side chains. The present paper presents a study of the quantitativeness of the alkaline borohydride procedure, using four parameters: the loss of seryl and threonyl residues, the formation of alanine and 2-aminobutanoic acid; the decrease in N-acetylhexosamine and the recovery of the amino sugar alcohols. Bovine, ovine and porcine submandibular glycoproteins were studied. Evidence is presented for the existence of N-acetylglucosamine involvement in O-glycosidic linkages to serine and threonine. Results for the relative rates of beta-elimination indicate that serine-linked glycosides are released more rapidly than threonine-linked glycosides. 相似文献
5.
K Watanabe 《Journal of biochemistry》1976,80(2):379-387
Mild alkaline treatment of glycopeptide (GP-II) resulted in the loss of 1 mole of serine and 5 moles of threonine per mole of GP-II, suggesting the presence of O-glycosyl bonds between 1 serine and 5 threonine residues and carbohydrate chains. Treatment of GP-II with alkaline borohydride released only disaccharide. Methylation studies of the carbohydrate moiety gave 2,3,4,6-tetra-O-methyl and 2,4,6-tri-O-methyl derivatives of mannose in a ratio of approximately 1:1. In addition, one step of Smith degradation resulted in the loss of about 6 residues of mannose per mole of GP-II. Moreover, alpha-mannosidase [EC 3.2.1.24] liberated about 6 residles of mannose per mole of GP-II. On the basis of these data, the structure of the carbohydrate moiety of GP-II was confirmed to be 3-O-alpha-mannosylmannose. The amino- and carboxyl-terminal amino acids of GP-II were determined to be threonine and serine, respectively. On reductive cleavage of N-proline bonds with metallic sodium in liquid ammonia, 2 moles of alanine per mole of GP-II were lost. From the compositions of three fragments isolated from the reductive cleavage products, the amino acid sequence of the peptide portion of GP-II was determined. Based on these data, a probable structure was proposed for GP-II. 相似文献
6.
M C Herzberg M J Levine S A Ellison L A Tabak 《The Journal of biological chemistry》1979,254(5):1487-1494
Highly purified mucin was prepared from monkey (Macaca arctoides) extraparotid saliva by sequential chromatography on Sephadex G-200 (followed by reduction and alkylation of void volume materials), Sepharose CL-2B with 6 M urea, and CM52 cellulose with 6 M urea. Purity was critically ascertained by anion exchange chromatography, ultracentrifugal analysis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide electrophoresis, and crossed immunoelectrophoresis. Use of crossed immunoelectrophoresis to examine mucin preparations has not been previously reported. This technique was useful for assessing purity and displaying charge and size microheterogeneity in the purified S-carboxymethylated mucin. Threonine and serine comprised 37.8% of the total amino acids while the oligosaccharide moiety contained N-acetyl-glucosamine, N-acetylgalactosamine, fucose, galactose, N-acetylneuraminic acid, and sulfate. Following alkaline borohydride treatment, the carbohydrate chains were found to be linked O-glycosidically between N-acetylgalactosamine and threonine (serine). 相似文献
7.
The chemical constitution of the carbohydrate portion of mycodextranase, an exocellular endo-alpha(1 yields 4) D-glucanase of Penicillium melinii, has been investigated. At least 80% of the carbohydrate, consisting exclusively of mannose and glucose, is released from protein by treatment of the enzyme with 0.05 M potassium hydroxide plus 1 M sodium borohydride or 0.5 M sodium hydroxide at 50 degrees. There is concomitant destruction of 60% of the threonine and 15% of the serine of the treated enzyme and an increase in absorption, at 241 nm, of the treated protein's spectrum, indicative of an O-glycosidic beta-hydroxyamino acyl linkage between untreated protein and its associated carbohydrate. Mannose is the monosaccharide involved in this linkage. Smith degradation, methylation, and glycosidase digestions of the carbohydrate indicate that it is present in mycodextranase as side chains of mannose, glucosyl alpha(1 yields 2)-mannose, and mannosyl alpha(1 yields 2)-glucosyl alpha(1 yields 2)-mannose units with each enzyme molecule bearing a calculated average of 25 side chains. Separation of pronase glycopeptides by gel filtration on Sephadex G-25 revealed that 96% of the carbohydrate is present in the highest molecular weight fraction which contains 60% of the threonine of mycodextranase but only 3.5% of the aromatic acids judged by its absorbance at 280 nm. Further fractionation of this glycopeptide component on Sephadex G-75 indicates carbohydrate is restricted to two fractions, one containing 71% by weight of the threonine and serine of mycodextranase and 56% of its carbohydrate. These results suggest carbohydrate chains of mycodextranase are clustered in a few threonin-rich regions along the polypeptide chain rather than being separated from each other by nonglycosylated areas. 相似文献
8.
Milk fat globule membrane was shown to contain sialic acid, all of which could be released without disruption of the fat globule. Sialoglycopeptides were cleaved from the surface of intact fat globules by Pronase and fractionated on Sephadex G-50. Further fractionation of the major sialoglycopeptide peak on DEAE-Sephadex gave two groups of sialoglycopeptides eluted with 0.1 M NaCl (Group A) and 0.5 M NaCl (Group B), respectively. Refractionation gave a major sialoglycopeptide from each of the two groups together with a total of three minor sialoglycopeptides. All five sialoglycopeptides eluted as single peaks using shallow salt gradients on DEAE-Sephadex and contained a hydrophilic peptide chain together with galactose, mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid. Glycopeptides of Group A but not Group B contained fucose. The major sialoglycopeptide of Group B released 35% of its hexose and hexosamine on treatment with alkaline borohydride leaving a sialoglycopeptide which had reduced serine and threonine and elevated alanine levels and in addition contained 2-aminobutyric acid. An oligosaccharide fraction containing N-acetylgalactosaminitol, galactose and sialic acid in a molar ratio of 1:1:2 was partially characterised from the clevage mixture. The major sialoglycopeptide of Group A had a more complex carbohydrate structure and showed no released carbohydrate on treatment with alkaline borohydride. The sialoglycopeptides of milk fat globule membrane show many similarities with those of erythrocyte membrane and have a potential use in comparative and structural studies. 相似文献
9.
The egg vitelline envelope of the marine invertebrate Megathura crenulata is a glycoprotein composed of 37.3 mol % protein and 62.7 mol % carbohydrate. Of the total amino acid content, 61 mol % consists of a single amino acid, threonine. The carbohydrate content includes galactosamine, galactose, and fucose. The molar ratio of threonine to galactosamine is about 1:1. Most of the threonine residues are linked to galactosamine residues via O-glycosidic bonds. A single peptide that was purified following alkaline borohydride treatment of the vitelline envelope had the structure: Abu-Pro-Abu-(Abu6, Pro1, Thr1), where Abu is 2-aminobutyric acid. Several sugar residues have been isolated following the alkaline hydrolysis of the vitelline envelope that include an octasaccharide Gal4Fu4, an hexasaccharide Gal3Fu3, a trisaccharide Gal3, fucose, and galactose. It is proposed that the vitelline envelope of Megathura crenulata eggs is composed of polypeptide chains built to a large extent of closely spaced threonine residues. Almost every threonine residue is linked to a saccharide moiety. 相似文献
10.
A high-molecular-weight glycoprotein has been purified from the cervical mucus of the bonnet monkey (Macaca radiata). The glycoprotein was shown to be homogeneous by electrophoresis, sedimentation equilibrium, and N-terminal group determination, and to contain 19% protein, 19% D-galactose, 18% N-acetyl-D-galactosamine, 15% N-acetyl-D-glucosamine, 11% L-fucose, 10% sialic acid, and 1% sulfate groups, corresponding to about 1800 amino acid residues and 400 carbohydrate side chains of about 9 monosaccharides. The carbohydrate chains are linked to the peptide backbone through N-acetyl-D-galactosamine and serine (or threonine) residues. Reduction with dithiothreitol and alkylation with iodoacetic acid reduced the molecular mass from 1 to 0.5 X 10(6) daltons and produced subunits having the same size, charge, and N-terminal amino acid. Electrophoretic studies suggested the presence of disulfide bonds between two chains of the glycoprotein. Degradation with alkaline borohydride gave, after fractionation on Bio-Gel P-2, fractions containing L-fucose, D-galactose, N-acetyl-D-galactosaminitol, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and sialic acid in the ratio of 1.0:3.0:1.0:1.0:1.3:1.0. Further fractionation by electrophoresis and paper chromatography gave a charged fraction representing 13% of the original glycoprotein. Enzymic degradation and methylation studies indicated the presence of the structure alpha-Gal-(1 leads to 3)-[Fuc(1 leads to 2)]-Gal-(1 leads to 4)-GlcNAc, linked to a core component containing N-acetyl-D-galactosaminitol. 相似文献
11.
Glycolipid antigens with blood group I and i specificities from human adult and umbilical cord erythrocytes 总被引:2,自引:0,他引:2
Y Okada R Kannagi S B Levery S Hakomori 《Journal of immunology (Baltimore, Md. : 1950)》1984,133(2):835-842
Neutral glycolipids and gangliosides of umbilical cord and adult human erythrocytes were separated by high performance liquid chromatography, and each fraction was analyzed by direct binding of anti-I (Ma) and anti-i (Den) on solid phase glycolipid-lecithin-cholesterol. The I- and i-active glycolipids were isolated and their structures were determined by methylation analysis and direct probe mass spectrometry. The major I antigen in adult erythrocytes, showing a remarkable binding activity with anti-I(Ma), was found in one neutral glycolipid fraction, designated fraction y4, which was identified as a mixture of two glycolipids of a new type, lactoisooctaosylceramide and monofucosyllactoisooctaosylceramide (for structures, see Table I). In addition, two gangliosides displaying direct binding activity with anti-I(Ma) were identified as monosialoganglioside G8, as previously described and disialosyllactoisooctaosylceramide, which showed the same level of I activity as the y4 glycolipid. The same ganglioside was recently isolated and characterized by Kundu and co-workers. The major i-active glycolipid antigen in umbilical cord erythrocytes, showing a strong binding activity with anti-i(Den), was a neutral glycolipid, x4a, which was identified as lactonorhexaosylceramide. This glycolipid without fucosyl or sialosyl substitution has not been isolated previously and was present as an obvious normal component of umbilical cord erythrocytes, but an extremely minor component of adult erythrocytes. Sialosyllactonorhexaosylceramide (G6) was isolated and characterized as a second i antigen of umbilical cord erythrocytes, but showed a very weak binding activity with the anti-i antibody. Although these sialosyl derivatives displayed only weak activity, the chemical quantity of the sialosyl derivatives is significantly large in fetal erythrocytes; therefore, Ii activity of human erythrocytes, in general, must be significantly dependent on sialosyl derivatives in addition to unsubstituted structures. 相似文献
12.
T Krusius J Finne R K Margolis R U Margolis 《The Journal of biological chemistry》1986,261(18):8237-8242
The chondroitin sulfate proteoglycan of rat brain was digested with Pronase, and after removal of glycosaminoglycans, the resulting glycopeptides were treated with alkaline borohydride to release O-glycosidically linked oligosaccharides. These were fractionated by ion exchange chromatography, gel filtration, and preparative thin layer chromatography, and their structural properties were studied by specific enzymatic degradations, methylation analysis, and gas-liquid chromatography-mass spectrometry of disaccharides as their trimethylsilylated and permethylated derivatives. In addition to the previously characterized N-acetyl-galactosamine-linked oligosaccharides and neutral mannitol-containing oligosaccharides [GlcNAc(beta 1-3) Manol and Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Manol] (where Fuc is fucose), we have now identified the sialylated tetrasaccharide NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc (beta 1-3)Manol, which accounts for approximately 20% of the mannitol-containing oligosaccharides. The proteoglycan also contains mannose-linked keratan sulfate chains (with a molecular size of 3,000 to 10,000 Da) composed of disaccharide repeating units consisting of Gal(beta 1-4)GlcNAc-6-O-SO4(beta 1-3), with a small proportion of branch points at C-6 of galactose residues. There is approximately one keratan sulfate chain per four chondroitin sulfate chains of 18,000-19,000 Da. After alkaline borohydride treatment of the neutral and monosialyl glycopeptide fractions, the combined decrease in mannose and N-acetylgalactosamine was very close to the observed destruction of serine + threonine and was accompanied by an equimolar increase in alanine and alpha-aminobutyric acid. One half of the mannose was destroyed by alkaline borohydride treatment of the glycopeptides and stoichiometrically converted to mannitol, while there were only small changes in the relative amounts of the other sugars and amino acids. The data demonstrate that over half of the carbohydrate-peptide linkages in the proteoglycan are of the mannosyl-O-serine/threonine type. 相似文献
13.
The pathogenic yeast Cladosporium werneckii produces a surface peptido-phosphogalactomannan (PPGM) with a peptide backbone rich in serine and threonine to which three types of carbohydrate chains are linked. These chains are: Type a, glactomannan units linked through phosphodiester bonds to produce long chains of molecular weight about 50,000; type b more numerous short mannosyl oligosaccharide units, and type c, more infrequent, long galactomannan chains. The first two are linked to the peptide through alkali-labile bonds to serine and threonine of the peptide whereas type c chains are linked through alkali-stable bonds (Lloyd, K. O. (1972) Biochemistry, 11, 3884–3890). The PPGM sample can be separated into three or four major components by diethylaminoethyl (DEAE-) Sephadex chromatography. By means of sequential degradations with alkali and acid, the structural basis for this heterogeneity has been demonstrated. It is due to the presence of different proportions of the three types of chains in the various fractions. The presence of O-acetyl groups, mainly on the a chains, was demonstrated in PPGM by chemical analysis and by proton and 13C nuclear magnetic resonance spectroscopy. Purified a chains were isolated from PPGM following Pronase digestion. By taking advantage of the alkali lability of the carbohydrate-protein linkages it has been possible to cleave the peptide moiety away from the carbohydrate. By sequential chromatography on Bio-Gel P-100, Dowex 1 and Bio-Gel P-100, five modified peptide fractions were isolated. The molecular weights of these fractions varied from 9,500 to 18,500 as judged by polyacrylamide-gel electrophoresis. Unlike the original peptide, the modified peptides contained low amounts of serine and threonine. The predominant amino acids were alanine, glycine, aspartic acid and glutamic acid which together make up between 51 and 55% of the peptides. The high content of the last two amino acids accounts for the acidic nature of the peptides. It appears that each fraction consists of a slightly heterogeneous population of peptides very similar in amino acid composition. It is not clear whether the compositional and size heterogeneity exists in the original peptide or whether it arose during the isolation procedure. 相似文献
14.
A A Preobrazhenski? L M Likhosherstov S N Senchenkova A I Rodionova S P Feshchenko 《Biokhimii?a (Moscow, Russia)》1989,54(8):1235-1246
Using immobilized monoclonal antibodies, a tissue-specific antigen, chordin, was isolated from cell extracts of giant sturgeon (beluga) notochord. The antigen was further purified by gel filtration through SP-Sephadex (pH 2.1) and gel chromatography on TSK Toyopearl HW-60. Purified chordin preparations contained 40% of protein and 60% of carbohydrates. The predominant polar amino acids were threonine, serine, glycine, asparagine and glutamine (or aspartic and glutamic amino acids). The carbohydrate moiety comprised mannose, fucose, galactose, galactosamine and glucosamine. Treatment of chordin with three enroglycosidases specifically hydrolyzing the carbohydrate chains of proteoglycans did not affect the antigenic properties of chordin or its behaviour on gel filtration. These findings and the fact that 75% of galactosamine was converted to galactosaminite after treatment with alkaline NaBH4 permitted to relate chordin to glycoproteins carrying O-glycosidic carbohydrate-peptide bonds between the N-acetyl-galactosamine and beta-hydroxyamino acid residues. Besides, chordin seems to contain a N-glycosylamide carbohydrate-peptide bond as can be judged from glucosaminite formation after treatment of the antigen with alkaline LiHB4. The changes in the antigenic properties of chordin after its treatment with neuraminidase, pronase, sodium periodate, alkali, alkaline NaBH4 or LiBH4 suggest that the polypeptide moiety of the chordin molecule and, perhaps, the N-acetylgalactosamine within the composition of the carbohydrate-peptide bond are involved in the construction of its most immunogenic determinants (P-determinants). 相似文献
15.
A glycoprotein has been found in normal serum which inhibits the hemagglutination caused by human anti-i cold agglutinins. This glycoprotein binds to a Sepharose anti-i affinity column at 4°C and can be quantitatively eluted at 37°C. The eluted glycoprotein is specific in that it has potent hemagglutination-inhibition activity against anti-i antibodies but does not inhibit the hemagglutination by anti-I, -A, -B, -H or by the lectins Con A, PHA or Vicia graminea. The anti-i affinity method should provide a simple procedure for large scale purification and subsequent characterization of the i-antigen. 相似文献
16.
Plasma membranes were isolated from an ascites hepatoma, AH 130, by the fluorescein mercuric acetate (FMA) method. Glycopeptides and mucopolysaccharides were prepared by digesting the membranes with pronase, then by fractionating the digest chromatographically and electrophoretically. Isolated fractions were analyzed for their amino acid and carbohydrate compositions. Results were compared with those for corresponding fractions from AH 66 (J. Biochem. 76, 319-333 (1974)). Mucopolysaccharides and a series of glycopeptides were isolated from the fraction excluded from Sephadex G-50. The mucopolysaccharides were identified as a family of heparan sulfates with different electrophoretic mobilities. The glycopeptides contained serine, threonine, galactose, galactosamine, glucosamine, and sialic acid as the major constituents as aspartic acid and mannose as minor ones. This suggests that most of the carbohydrate moieties are linked to serine or threonine (O-glycosidic), and that some are linked to asparagine (N-glycosidic). No nearly purely O-glycosidic glycopeptides were found in this fraction from AH 130, through they were the major glycopeptides from the AH 66 plasma membranes. In the fraction included in the gel, glycopeptides containing fucose, galactose, mannose, glucosamine, glaactosamine, and sialic acid were found. The presence of galactosamine suggests that some of the glycopeptides are O-glycosidic though most are N-glycosidic. In the corresponding fraction from AH 66, nearly purely N-glycosidic glycopeptides were found. 相似文献
17.
Milk fat globule membrane was shown to contain sialic acid, all of which could be released without disruption of the fat globule. Sialoglycopeptides were cleaved from the surface of intact fat globules by Pronase and fractionated on Sephadex G-50. Further fractionation of the major sialoglycopeptide peak on DEAE-Sephadex gave two groups of sialoglycopeptides eluted with 0.1 M NaCI (Group A) and 0.5 M NaCI (Group B), respectively. Refractionation gave a major sialoglycopeptide from each of the two groups together with a total of three minor sialoglycopeptides. All five sialoglycopeptides eluted as single peaks using shallow salt gradients on DEAE-Sephadex and contained a hydrophilic peptide chain together with galactose, mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid. Glycopeptides of Group A but not Group B contained fucose.The major sialoglycopeptide of Group B released 35 % of its hexose and hexosamine on treatment with alkaline borohydride leaving a sialoglycopeptide which had reduced serine and threonine and elevated alanine levels and in addition contained 2-aminobutyric acid. An oligosaccharide fraction containing N-acetylgalactosaminitol galactose and sialic acid in a molar ratio of 1 : 1 : 2 was partially characterised from the cleavage mixture.The major sialoglycopeptide of Group A had a more complex carbohydrate structure and showed no released carbohydrate on treatment with alkaline borohydride.The sialoglycopeptides of milk fat globule membrane show many similarities with those of erythrocyte membrane and have a potential use in comparative and structural studies. 相似文献
18.
Immunochemical studies on the specificity of the peanut (Arachis hypogaea) agglutinin. 总被引:6,自引:0,他引:6
The specificity of purified, peanut agglutinin has been studied immunochemically by quantitative precipitin and inhibition assays. The lectin showed substantial differences in precipitating with blood-group substances of the same specificity. Of the B substances tested, horse 4 25% completely precipitated the lectin, Beach phenol insoluble failed to interact, and PM phenol insoluble gave an intermediate reaction. The lectin did not precipitate with A1 substances, with hog gastric mucin A + H substance, or with A2 substance WG phenol insoluble. Another A2 substance, cyst 14 phenol insoluble, precipitated approximately 2/3 of the lectin. Of the H substances, Tighe phenol insoluble was inactive, JS phenol insoluble precipitated poorly, and morgan standard H precipitated about 80% of the lectin. However, first stage of Smith degradation, as well as Pl fractions obtained by mild acid hydrolysis of blood-group substances, gave products which precipitated strongly. The lectin was also completely precipitated by all precursor blood-group substances, as well as by cows 21 and 26, all having strong I-Ma, I-Ort, I-Step, and I-Da activities. Cow 18, which does not possess significant blood-group I activity, precipitated very slightly. Fractions of blood-group substances N-1 (Lea) and Tij (B) obtained by precipitation from 90 percent phenol at higher concentrations of ethanol interacted better with peanut agglutinin. These differences in activity are ascribable to a heterogeneity resulting from incomplete biosynthesis of carbohydrate side-chains of blood-group substances, particularly resulting in variations in the numbers of DGalbeta1 leads to 3DGalNAc or DGalbeta1 leads to 4DGlcNAc determinants. The agglutinin reacted with the hydatid cyst P1 glycoprotein, as well as with the previously studied antifreeze and sialic acid-free alpha1 acid glycoproteins, but not with pneumococcus type XIV polysaccharide. Inhibition of precipitation showed the lectin to be most specific for the disaccharide DGalbeta1 leads to 3DGalNAc, which is 14, 55, and 90 times as active as DGalbeta1 leads to 4DGlcNAc, DGal, and DGalbeta1 leads to 3DGlcNAc, respectively. DGalbeta1 leads to 3N-acetyl-D-galactosaminitol has approximately 1/25th the activity of DGalbeta1 leads to 3DGalNAc. Substitutions of DGlcNAc or LFuc on the DGal of active inhibitors completely blocked the activity, in line with the assumption that the combining site of the peanut lectin is a partial cavity. The oligosaccharides DGalbeta1 leads to 4DGlcNAcbeta1 leads to 6-hexane-1,2,4,5,6-pentol(s) and DGalbeta1 leads to 3[DGalbeta1 leads to 4DGlcNAcbeta1 leads to 6]N-acetyl-D-galactosaminitol showed the same inhibitory activity as DGalbeta1 leads to 4DGlcNAc, suggesting that the combining site of the peanut agglutinin may not be complementary to more than a disaccharide... 相似文献
19.
Intima-media of porcine thoracic aorta were digested with pronase, after extraction of the saline-soluble matters and fat. A glycopeptide fraction was precipitated with 90% (vol/vol) ethanol from the 80% ethanol-soluble fraction of the trichloroacetic acid (7%)-soluble fraction of the pronase digest. The glycopeptide fraction was fractionated by affinity chromatography on concanavalin A (Con A)-Sepharose 4B, yielding 4 fractions (FA, FB, FC and FD). The most carbohydrate-rich fraction (FB) was further purified to a homogeneous state. The purified FB (FB-0.1) and all other fractions contained desmosine and isodesmosine. The major sugars in the fractions without or with low affinity for Con A (FA, FB, and FB-0.1) were glucosamine, galactose, mannose and sialic acid, while those in the fractions with high affinity for this lectin (FC and FD) were glucosamine, glucose and mannose. All the fractions contained glycine, aspartic acid (and/or asparagine), serine, proline, threonine, glutamic acid (and/or glutamine) and alanine as the major amino acids, amounting to approximately 80% of the total. 相似文献
20.
S Bjoern D C Foster L Thim F C Wiberg M Christensen Y Komiyama A H Pedersen W Kisiel 《The Journal of biological chemistry》1991,266(17):11051-11057
Factor VII is a multidomain, vitamin K-dependent plasma glycoprotein that participates in the extrinsic pathway of blood coagulation. Earlier studies demonstrated a novel disaccharide (Xyl-Glc) or trisaccharide (Xyl2-Glc) O-glycosidically linked to serine 52 in human plasma factor VII (Nishimura, H., Kawabata, S., Kisiel, W., Hase, S., Ikenaka, T., Shimonishi, Y., and Iwanaga, S. (1989) J. Biol. Chem. 264, 20320-20325). In the present study, human plasma and recombinant factor VII were isolated and subjected to enzymatic fragmentation. Peptides comprising residues 48-62 of the first epidermal growth factor-like domain of each factor VII preparation were isolated for comparative analysis. Using a combined strategy of amino acid sequencing, carbohydrate and amino acid composition analysis, and mass spectrometry, three different glycan structures consisting of either glucose, glucose-xylose, or glucose-(xylose)2 were detected O-glycosidically linked to serine 52 in plasma and recombinant factor VII. Approximately equal amounts of the three glycan structures were observed in plasma factor VII, whereas in recombinant factor VII the glucose and the glucose-(xylose)2 structures predominated. In addition to the O-linked glycan structures observed at serine 52, a single fucose was found to be covalently linked at serine 60 in both human plasma and recombinant factor VII. Carbohydrate and mass spectrometry analyses indicated that the fucosylation of serine 60 was virtually quantitative. Metabolic labeling studies using [14C]fucose confirmed the presence of O-linked fucose at serine 60. In order to assess whether the carbohydrate moiety at serine 52 contributes to the biological activity of factor VII, we have constructed a site-specific mutant of recombinant factor VII in which serine 52 has been replaced with an alanine residue. Mutant factor VIIa exhibited approximately 60% of the coagulant activity of wild-type factor VIIa in a clotting assay. The amidolytic activity of mutant factor VIIa was indistinguishable from that observed for recombinant wild-type factor VIIa. In addition, the ability of mutant factor VIIa in complex with either purified relipidated tissue factor apoprotein or tissue factor on the surface of a human bladder carcinoma cell line (J82) to activate either factor X or factor IX was virtually identical to that observed for wild-type factor VIIa. These results indicate that the carbohydrate moiety O-glycosidically linked to serine 52 does not appear to be involved either in the interaction of factor VIIa with tissue factor, or the expression of its proteolytic activity toward factor X or factor IX following complex formation with tissue factor.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献