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1.
A receptor glycopeptide for concanavalin A was isolated from calf thymocytes by a method originally devised for the isolation of a lectin receptor from human erythrocytes (Kubánek, J., Entlicher, G.; and Kocourek, J. [1973] Biochim, Biophys. Acta 304, 93–102). The method consisted of pronase digestion of the lipid-depleted thymocyte membrane material followed by ethanol fractionation, separation on Sephadex and preparative paper electrophoresis. The isolated glycopeptide contains 10.4% of neutral sugar. The molar ratios of the sugar components mannose, galactose, glucosamine, glucose, fucose and sialic acid are 3 : 2 : 2 : 1 : 1 : 1. The minimum molecular weight calculated from the sugar composition is about 12 000.Concanavalin A receptor activity of the glycopeptide was demonstrated in three different ways: (i) Reduction of the 125I-labeled concanavalin A binding to thymocytes. (ii) Prevention of concanavalin A induced agglutination of calf thymocytes. (iii) Inhibition of concanavalin A stimulated DNA synthesis in calf and rabbit thymocytes and rabbit lymph node lymphocytes cultivated in vitro.The isolated glycopeptide seems to be involved in the interaction of lymphocytes with concanavalin A and the subsequent stimulation.  相似文献   

2.
Myelin was purified from rat brain and sciatic nerve after invivo labeling with [3H]fucose and [14C]glucosamine to provide a radioactive marker for glycoproteins. The glycoproteins in the isolated myelin were digested exhaustively with pronase, and glycopeptides were isolated from the digest by gel filtration on Bio-Gel P-10. The glycopeptides from brain myelin separated into large and small molecular weight fractions, whereas the glycopeptides of sciatic nerve myelin eluted as a single symmetrical peak. The large and small glycopeptide fractions from central myelin and the single glycopeptide fraction from peripheral myelin were analyzed for carbohydrate by colorimetric and gas liquid chromatographic techniques. The glycopeptides from brain myelin contained 2.4 μg of neutral sugar and 0.59 μg of sialic acid per mg total myelin protein, whereas sciatic nerve myelin glycopeptides contained 10 μg of neutral sugar and 3.8 μg of sialic acid per mg total protein. Similarly, the gas-liquid chromatographic analyses showed that the glycopeptides from peripheral myelin contained 4- to 7-fold more of each individual per mg total myelin protein than those from central myelin. Most of the sialic acid and galactose in the glycopeptides from central myelin were in the large molecular weight fraction, and the small molecular weight glycopeptides contained primarily mannose and N-acetylglucosamine. The considerably higher content of glycoprotein-carbohydrate in peripheral myelin supports the results of gel electrophoretic studies, which indicate that the major protein in peripheral myelin in glycosylated while the glycoproteins in purified central myelin are quantitatevely minor components.  相似文献   

3.
From 1 kg of dried Ononis hircina Jacq. roots 36 mg of a lectin were isolated by affinity chromatography on O-β-lactosyl polyacrylamide gel. The lectin is homogeneous as judged by ultracentrifugal analysis (s20,w = 6.2 S), polyacrylamide disc electrophoresis at pH 8.9 or 4.5, gel filtration on thin layers of Sephadex G-200 (Mr = 110 000) and dodecyl sulfate electrophoresis (Mr of sub-units 31 000, both in presence and absence of mercaptoethanol) and disc dodecyl sulfate electrophoresis (pH 9.5). The lectin contains much aspartic and glutamic acids, serine and threonine and also 7.2% of neutral sugar. It is relatively specific for human type O erythrocytes that are agglutinated at a minimal lectin concentration 0.3 μg/ml. The erythroagglutinating activity is not stimulated by Ca2+, Zn2+, Mg2+, Mn2+, Co2+, or Ni2+ salts; it is inhibited most effectively by N-acetyl-D-galactosamineandanumberofD-galactosederivatives. Dissociation constants of several lectin · sugar complexes were estimated by affinity electrophoresis. The lectin is not mitogenic in rabbit lymph nodes lymphocytes.  相似文献   

4.
Semliki Forest virus was grown in BHK cells and labeled in vivo with radio-active monosaccharides. promnase digenst of the virus chromatographer on Bio-Gel P 6 revealed glycopeptides of A-type and B-type. (For the nomenclature see Johnson J. and Clamp J.R. (1971) Biochem. J. 123, 739–745) The former was labeled with [3H]fucose, [3H]galactose, [3H]mannose and [14C]glucosamine, the latter only with [3H]mannose and [14C]glucosamine. The three envelope glycoproteins E1, E2 and E3 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to pronase digestion. The glycoproteins E1 and E3 revealed glycopeptides of A-type. E2 revealed glycopeptides of B-type. E2 yielded additionally a glycopeptide (Mr3100) which was heavily labeled from [3H]galactose, but only marginally from [14C]glucosamine, [3H]fucose and [3H]mannose. Wether this glycopeptide belongs to the A-type or not remains uncertain. The apparent molecular weights of the A-type units measured by gel filtration were 3400 in E1 and 4000 in E3; the B-type unit of E2 had an apparent molecular weight of 2000. Combined with the findings of our earlier chemical analysis these data suggast that E1 and E3 contain on the average one A-type unit; E2 probably contains one 3100 dalton unit plus one or two B-type units.  相似文献   

5.
TheN-linked oligosaccharides of cell-CAM 105, a glycoprotein involved in the intercellular adhesion between rat hepatocytes, were studied by sequential lectin-agarose affinity chromatography of desialylated, [14C]-labelled glycopeptides. These glycopeptides were obtained by extensive pronase digestion followed byN-[14C]acetylation of the peptide moieties and desialylation by mild acid hydrolysis.Assuming that all glycopeptides were radiolabelled to the same specific radioactivity, Concanavalin A-Sepharose chromatography indicated that the majority of the glycans (84%) were of the complex-type of which approximately half were bi-antennary structures. The remainder of the glycans comprised oligomannose-type structures and/or incomplete bi-antennary structures.Pisum sativum lectin-agarose chromatography revealed that part of the bi-antennary glycans contained a fucose residue (1-6)-linked to theN-acetylglucosamine which is attached to asparagine. Furthermore, the presence of tri-, and tetra- and/or tri'-antennary complex-type glycans was demonstrated by chromatography on immobilizedPhaseolus vulgaris leukoagglutinating phytohemagglutinin andAleuria aurantia lectin (AAL). AAL-agarose chromatography furthermore indicated the presence of (1-3)-linked fucose in part of these glycopeptides, whereas no (1-6)-linked fucose could be detected in these structures.The degree of -galactosylation of the complex-type glycans was investigated by chromatography onRicinus communis agglutinin-agarose. The results indicated that only part of the bi-antennary glycans were completely -galactosylated. Similarly, at least three -galactose residues were present in only a part of the tri-, and tetra- and/or tri'-antennary glycans.Abbreviations CAM cell adhesion molecule - ConA Concanavalin A - WGA wheat germ agglutinin - PEA Pisum sativum lectin - E-PHA Phaseolus vulgaris erythroagglutinating phytochemagglutinin - L-PHA Phaseolus vulgaris leukoagglutinating phytohemagglutinin - RCA Ricinus communis agglutinin 1 - AAL Aleuria aurantia lectin - mGlc methyl--d-glucopyranoside - mMan methyl--d-mannopyranoside - CO, WO, PO, EO, LO, RO, AO, nonretained, and Cn, Wn, Pn, En, Ln, Rn, An (n=1–4) retarded or bound glycopeptide fractions on columns of immobilized ConA, WGA, PEA, E-PHA, L-PHA, RCA, and AAL, respectively The fraction names are also used sequentially, e.g. C1P1, which indicates the fraction of glycopeptides that was eluted from ConA-Sepharose on position C1 and was subsequently eluted from PEA-agarose on position P1.  相似文献   

6.
《Phytochemistry》1986,25(2):323-327
A lectin has been purified from L. capassa seed by ammonium sulphate fractionation and affinity chromatography on a column of D-galactose-derivatized Sepharose. The lectin is a glycoprotein which contains 3.8% neutral carbohydrates comprised of mannose, N-acetylglucosamine, xylose and fucose. The subunit M, of the lectin is 29 000, it has only alanine as N-terminal amino acid and contains 240 amino acids with a high content of acidic and hydroxy amino acids, single residues of methionine and histidine and the absence ofcystine. The lectin of L. capassa seed is a metalloprotein in that it contains 0.8 mol Ca2+ and 0.4 mol Mn2+ per mol. It agglutinates untreated human A, O and B type erythrocytes and rabbit erythrocytes. N-Acetyl-D-galactosamine was the best inhibitor. D-Galactose and various carbohydrates containing this sugar inhibit the hemagglutinating activity of the lectin. The lectin is also inhibited by D-glucose. The amino-terminal sequence of the lectin from L. capassa seed shows a significant degree of homology with many lectins from leguminous plants and is related to concanavalin A by a circularly permuted sequence homology.  相似文献   

7.
Cultures of soybean cells incorporate [5,6-3H]-l-fucose into various cellular components including lipids and proteins. The membrane glyco-proteins were digested with pronase to produce glycopeptides, and the glycopeptides were isolated on columns of Biogel P-4. The major fucoselabeled glycopeptide sized as a Hexose15-17-N-acetylglucosamine2 (GlcNAc2) on columns of Biogel P-4. Fucose incorporation was also examined in the presence of the processing inhibitor swainsonine, and the glycosylation inhibitor tunicamycin. In the presence of swainsonine, the incorporation of fucose was not reduced but the glycopeptides were smaller in size and migrated like Hexose12-13-GlcNAc2 structures. On the other hand, tunicamycin inhibited the incorporation of fucose into the glycopeptides by 70 to 80%, indicating that the l-fucose was present in N-linked oligosaccharides.  相似文献   

8.
From cells of a nullipotential line of embryonal carcinoma was isolated a membrane fraction enriched in the cell surface F9 antigen. More than 40% of the radioactive fucose and galactose incorporated by cells into nondialyzable material was recovered in this membrane preparation, corresponding to an approximately 10-fold purification of the labeled material. Extreme heterogeneity of membrane glycoproteins labeled with these sugars was revealed by sodium dodecyl sulfate gel electrophoresis. Glycopeptides prepared by extensive pronase digestion of membranes labeled with fucose or galactose showed properties similar to those already described for fucose-labeled glycopeptides from whole cells. Namely, large glycopeptides eluted near the excluded volume of Sephadex G-50 column were the predominant glycopeptide species, while complex glycopeptides of molecular weight around 2500 were minor components. Therefore, these large glycopeptides, characteristic of embryonal carcinoma cells, are derived mainly from a variety of glycoproteins closely associated with the membrane system, most probably cell-surface membrane of the cells. The large glycopeptides were also significantly labeled with glucosamine, but only slightly with mannose; major components of mannose-labeled glycopeptides from the membranes were high-mannose glycopeptides of low molecular weight. Several experiments excluded the possibility that the larg glycopeptides are mucopolysaccharides, glycolipids or mucin-type glycoproteins with short oligosaccharide chains.  相似文献   

9.
The in vivo incorporation of [3 5S]sulfate and [3H]fucose into rat brain myelin was investigated. Most of the 3 5S in the myelin was in sulfatide, but about 4% was associated with the residual proteins after chloroform/methanol extraction. Polyacrylamide gel electrophoresis of these proteins indicated that the major 3 5S-labeled component corresponded to the major fucose-labeled glycoprotein. The labeling of this predominant glycoprotein with sulfate was more selective than with fucose, since there was relatively little incorporation of sulfate into some of the minor fucose-labeled glycoproteins. There was little or no 3 5S associated with proteolipid or basic protein on polyacrylamide gels. The fucose-labeled glycoproteins were converted to glycopeptides by pronase digestion and separated into two major classes by gel filtration on Sephadex-G50. Only the higher molecular weight class contained significant amounts of 3 5S. The association of 3 5S with the glycopeptides was not due to binding of sulfatide or free inorganic sulfate. The results indicate that the predominant myelin-associated glycoprotein in rat brain is sulfated.  相似文献   

10.
Three cell surface components of mouse embryonal carcinoma (EC) cells, F9 antigens and the receptors to the lectins FBP and PNA, have been isolated from radiolabeled EC cells by indirect immunoprecipitation. All three were efficiently labeled with fucose, galactose and glucosamine, but scarcely at all with mannose. The high molecular weight glycopeptides characteristic of early embryonic cells were released as the major glycopeptides upon pronase digestion of the three markers. The binding sites to the two lectins are present in the high molecular weight glycopeptides. Furthermore, a close correlation exists between the disappearance of the high molecular weight glycopeptides from differentiating EC cells and the disappearance of the three markers from the surface of these cells. The large glycopeptides from the three markers have the following properties in common. First, they are not mucin-type glycopeptides with short oligosaccharides, glycolipids and acidic mucopolysaccharides, nor are they products of incomplete pronase digestion of conventional complex-type glycopeptides. Second, they do not contain appreciable amounts of Fucα1→2Gal or Fucα1→6GlcNAc linkages. Third, a significant fraction of the glycopeptides have the GlcNAcβ→Gal sequence in their core structure. We propose that the cell surface markers of EC cells have a class of large carbohydrate chains not found in typical surface markers of adult cells such as H-2, la and LETS.  相似文献   

11.
Glycosylated compounds associated with the carbohydrate-rich tubular membrane system of the oxyntic cell were investigated. Two glycopeptide fractions, designated Peaks A and B, were isolated from pronase digests of bullfrog oxyntic cell microsomes. Molecular sieve chromatography and cellulose acetate electrophoresis revealed that, although somewhat heterogeneous, each peak was composed primarily of glycopeptides with similar molecular weights and net charge densities. Peak B glycopeptides had a mean molecular weight of about 6000 and contained 70% of the recovered carbohydrate in the following molar ratios: hexose, 1.00; N-acetylhexosamine, 0.71; fucose, 0.61; sialic acids, <0.03. Peak a glycopeptides were considerably larger (molecular weight approx. 100 000) and contained carbohydrates in molar ratios similar to those of Peak B. In both peaks galactose and N-acetylglucosamine, respectively, were the predominant hexose and amino sugar isomers.The glycolipid content of bullfrog oxyntic cell microsomes was assessed by qualitative and quantitative thin-layer chromatography. The most abundant glycolipids were monoglucosylceramides (0.098 mole/mole phospholipid) and monogalactosylceramides (0.046 mole/mole phospholipid). Small quantities of sulfatides and gangliosides were also present.A compilation of available data regarding the chemical composition of the microsomes revealed that these membranes resemble plasma membranes in having high molar ratios of cholesterol to phospholipid (approx. 1.0) and large quantities of carbohydrate (225 μg/mg protein). The possible significance of these compositional features in protecting the oxyntic cell is discussed.  相似文献   

12.
Three glycopeptides were isolated from the pronase digest of the protein moiety of pig serum low density lipoprotein. The isolation procedure consisted of pronase digestion, gel filtration on Sephadex G-25 and G-50 columns, paper chromatography and DEAE-Sephadex A-50 column chromatography. Based on the carbohydrate analysis, the isolated glycopeptides were classified into two types. One type (GDI) consisted of mannose and N-acetylglucosamine residues in the molar ratio of 6:2 and had a molecular weight of about 2,300. The other type (GDII and GDIII) consisted of sialic acid, mannose, galactose, fucose, and N-acetylglucosamine residues in the molar ratio of 1:4:2:1:3 and 2:4:3:1:3, respectively. The molecular weights of GDII and GDIII were about 2,100 and 3,100, respectively. The results on the strong alkaline treatment of these glycopeptides suggested that all carbohydrate chains were linked to the peptide chains through N-acetylglucosaminyl-asparagine linkages. Of these glycopeptides and pig serum lipoproteins, only glycopeptide GDI and native LDL strongly interacted with concanavalin A.  相似文献   

13.
The lipid-free protein residue of rat brain tissue was treated with papain to solubilize the heteropolysaccharide chains of the tissue glycoproteins. The glycopeptides were separated into non-dialyzable and dialyzable glycopeptide preparations. Each preparation was then sorted out into groups of glycopeptides by means of electrophoresis and gel filtration. The quantitatively predominant glycopeptides were the alkali-stable glycopeptides (Group A) which accounted for 64% of the glycopeptide carbohydrate recovered from rat brain. Most of the group A glycopeptides appeared in the non-dialyzable preparation. The molecular weight of the glycopeptides of Group A ranged from approximately 5200–3700. The largest glycopeptide molecule in this mixture possessed the highest electrophoretic mobility and contained one fucose, four N-acetylneuraminic acid (NANA), six N-acetylglucosamine, four galactose, and three mannose residues per molecule. The spectrum of glycopeptides isolated in this group showed a progressive decrease in NANA rsidues, NANA and galactose residues, and NANA, galactose, and N-acetylglucosamine residues which could be correlated with a progressive decline in molecular weight and electrophoretic mobility. Some of the glycopeptides in each fraction recovered from this group of glycopeptides contained sulfate ester groups.A second group of glycopeptides (Group C glycopeptides) accounted for 25% of the total glycoprotein carbohydrate recovered from rat brain. These were recoverd from the dialyzable glycopeptide preparation, and resolved into three fractions by column electrophoresis. These glycopeptides do not contain sulfate, are composed predominately of mannose and N-acetylglucosamine, and possess a molecular weight of approximately 3000.Several minor groups of glycopeptides were detected. Alkali-labile glycopeptides (Group B) appeared in the non-dialyzable glycopeptide preparation. The dialyzable glycopeptide preparation contained glycopeptides (Group E) which contained N-acetylgalactosamine and glucose. These had a molecular weight of approximately 2000. Group D glycopeptides recovered from the dialyzable glycopeptide preparation contained variable amounts of NANA, mannose, galactose, N-acetylglucosamine, and sulfate. These possessed a molecular weight of approximately 2900.  相似文献   

14.
Monosaccharide Sequence of Protein-Bound Glycans of Uukuniemi Virus   总被引:3,自引:10,他引:3       下载免费PDF全文
Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells in the presence of [3H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [3H]mannose-labeled glycopeptides of the mixture of the two envelope glycoproteins G1 and G2 were characterized by degrading the glycans with specific exo-and endoglycosidases, by chemical methods, and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into three categories: complex, high-mannose type, and intermediate. The complex glycopeptides probably contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core (Man)3(GlcNAc)2 peptide. The high-mannose-type glycans were estimated to contain at least five mannose units attached to two N-acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The results suggested that the intermediate glycopeptides contained a mannosyl core. In about half of the molecules, one branch appeared to be terminated in mannose, and one appeared to be terminated in N-acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. Electron microscopic data suggest that the virions bud into smooth-surfaced cisternae in the Golgi region. The relative amounts of [3H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62, and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated [35S]methionine-labeled G1 and G2 proteins suggested that most of the complex and intermediate chains were attached to G1 and that most of the high-mannose-type chains were attached to G2.  相似文献   

15.
The oligosaccharide structures ofCry j I, a major allergenic glycoprotein ofCryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz1H-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch.Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides asCry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing 1–6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The cross-reactivities of human allergic sera to miraculin andClerodendron Trichotomum lectin (CTA) were low, and inhibition studies suggested that the oligosaccharides onCry j I contribute little or only conformationally to the reactivity of specific IgE antibodies.Abbreviations Cry j I a major allergenic glycoprotein ofCryptomeria japonica - B-SJA-II Sophora japonica bark lectin II - CTA Clerodendron trichotomum lectin - TFMS trifluoromethanesulfonic acid - HRP horseradish peroxidase  相似文献   

16.
Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with α-methylmannoside, constitute about 25–30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chrolide columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with α-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.  相似文献   

17.
Thyrotropin (TSH) is a glycoprotein hormone whose secretion from the anterior pituitary is regulated, in part, by the hypothalamic tripeptide thyrotropin-releasing hormone (TRH). We have used serial lectin affinity analysis to explore whether TRH, in addition to promoting TSH secretion, alters the carbohydrate structure of secreted TSH. Hypothyroid mouse hemipituitaries were incubated in medium containing [3H] mannose, [3H]glucosamine, or [3H]fucose either with or without 10(-7) M TRH. TSH was immunoprecipitated, proteolytically digested into glycopeptides, and chromatographed on serial lectin-Sepharose columns. Under basal conditions, 37% of secreted [3H]mannose-labeled TSH glycopeptides failed to bind to concanavalin A (ConA)-Sepharose, 55% bound and eluted with 10 mM alpha-methylglucoside, and 8% bound and eluted with 500 mM alpha-methylmannoside. Approximately 35% of glycopeptides not binding to ConA-Sepharose were bound by pea lectin-Sepharose, suggesting the presence of certain core fucosylated triantennary complex oligosaccharides. TRH caused a 2-fold increase in secretion of [3H]mannose-labeled TSH glycopeptides due almost exclusively to a specific increase in structures that bound to ConA-Sepharose and eluted with 10mM alpha-methylglucoside, corresponding to biantennary complex or unusual hybrid species. There was no change in the distribution of intrapituitary TSH glycopeptides with TRH. Acid hydrolysis of secreted proteins showed little metabolism of the tritiated sugar precursors, except for a 20% conversion of [3H]mannose to [3H]fucose. Moreover, ConA-Sepharose chromatography of secreted [3H]glucosamine- and [3H]fucose-labeled TSH glycopeptides showed similar increases in ConA-Sepharose binding with TRH as noted with [3H]mannose labeling. Subsequent lectin analysis of secreted [3H] mannose-labeled TSH glycopeptides on erythroagglutinating phytohemagglutinin-Sepharose and leukoagglutinating phytohemagglutinin-Sepharose disclosed no significant differences in TRH-treated versus control samples. These data suggest that secreted mouse TSH has greater carbohydrate heterogeneity than has been recognized previously. In addition, TRH in vitro promotes the secretion of specific TSH molecules apparently enriched in biantennary complex or unusual hybrid oligosaccharides.  相似文献   

18.
Semliki Forest virus was grown in BHK cells and labeled in vivo with radioactive monosaccharides. Pronase digests of the virus chromatographed on Bio-Gel P6 revealed glycopeptides of A-type and B-type. (For the nomenclature see Johnson, J. and Clamp, J.R. (1971) Biochem. J. 123, 739-745.) The former was labeled with [3H]fucose, [3H]galactose, [3H]mannose and [14C]glucosamine, the latter only with [3H]mannose and [14C]glucosamine. The three envelope glycoproteins E1, E2 and E3 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to pronase digestion. The glycoproteins E1 and E3 revealed glycopeptides of A-type. E2 revealed glycopeptides of B-type. E2 yielded additionally a glycopeptide (Mr3100) which was heavily labeled from [3H]galactose, but only marginally from [14C]glucosamine, [3H]fucose and [3H]mannose. Whether this glycopeptide belongs to the A-type or not remains uncertain. The apparent molecular weights of the A-type units measured by gel filtration were 3400 in E1 and 4000 in E3; the B-type unit of E2 had an apparent molecular weight of 2000. Combined with the findings of our earlier chemical analysis these data suggest that E1 and E3 contain on the average one A-type unit; E2 probably contains one 3100 dalton unit plus one or two B-type units.  相似文献   

19.
Glycopeptides were isolated from the surface of human diploid cells maintained strictly in the growing, and non-growing, state by daily feeding at optimum pH. By use of extensive pronase and endoglycosidase (Muramatsu, 1971) digestion we reduced surface glycopeptides to a fragment containing only a few amino acids and sugar moieties. We report a growth-dependent alteration near the carbohydrate-peptide linkage region. The heterogeneous glycopeptide fragments representing this region have an average molecular weight of approximately 800. Partial characterization of these fragments show that they contain fucose in α-linkage, hexosamine and aspartic acid.  相似文献   

20.
We have studied the synthesis of protein-bound carbohydrates in differentiating male germ cells in the mouse. Spermatocytes and spermatids synthesize asparagine-linked and high-molecular-weight glycopeptides as the major classes of protein bound carbohydrates. Asparagine-linked glycopeptides were found to be mainly composed of the complex bi-antennary type as shown by affinity chromatography on concanavalin-A Sepharose; high-molecular-weight glycopeptides were represented by nonfucosylated lactosaminoglycans since they were metabolically labeled with [14C]glucosamine but not with [3H]fucose, did not bind to DEAE-cellulose, and were susceptible to endo-β-galactosidase. Labeling with galactose oxidase/Na B3H4 technique demonstrated that lactosaminoglycans were present on the surface of differentiating germ cells and of testicular and epididymal spermatozoa. Since lactosaminoglycans from germ cells and testicular spermatozoa were not retained on a column of fucose-binding lectin, it was concluded that these molecules do not contain fucose. On the other hand, epididymal spermatozoa lactosaminoglycans bound to the lectin and therefore contained fucose. A soluble fucosyltransferase, capable of transferring fucose to germ cell lactosaminoglycans, was found to be present in the epididymis but not in the testis. These data show that developing germ cells synthesize nonfucosylated lactosaminoglycans which are probably preserved throughout spermiogenesis. We suggest that these molecules are fucosylated in vivo by a fucosyltransferase secreted by the epididymal epithelium.  相似文献   

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