A new glycopeptide was isolated from the glomerular basement membrane (GBM) of normal rats. Unlike already known glycopeptides, this glycopeptide has biological activity (nephritogenic activity) to induce glomerulonephritis when injected once into the footpads of homologous animals. A close relationship was found between the nephritogenic activity and the non-dialyzable glucose content of this glycopeptide. Thus the nephritogenic activity can be assessed quantitatively by estimating the content of "non-dialyzable glucose." Chemical purification of the nephritogenic glycopeptide involved the selective removal of inactive glycopeptide containing galactose, mannose, and N-acetyl-glucosamine (but no glucose). Trichloroacetic acid (TCA) treatment was a simple but highly effective procedure for selective removal of this inactive glycopeptide. The non-reducing terminus of the nephritogenic glycopeptide is alpha-D-glucopyranoside, and the glycopeptide reacts specifically with concanavalin A, even in the crude state. We propose that the nephritogenic glycopeptide is not an artifact produced during exhaustive proteolytic digestion, but a natural substance having a fixed molecular shape, even in the crude state, and whose union with GBM-proper can be easily broken by proteolytic digestion. 相似文献
A method for isolation of a potent nephritogenic antigen from bovine glomerular basement membrane has been established; the glomerular basement membrane was solubilized by trypsin digestion and fractionated successively by gel filtration on Ultrogel AcA-34, concanavalin A affinity chromatography and affinity chromatography on immobilized antibodies. The antigen thus prepared was found to be highly nephritogenic; it causes glomerulonephritis in rats by a single injection of 0.1 mg per individual. Amino acid and carbohydrate analyses revealed that the antigen is a glycoprotein which contains amino acids and sugars characteristic of collagen, namely, hydroxyproline, hydroxylysine, glycine, glucose and galactose, although the relative amounts of these amino acids and sugars are less than those found in Type IV collagen of glomerular basement membrane. 相似文献
1. The structure of the carbohydrate component of the glycopeptide isolated from the proteolytic digest of ovalbumin has been investigated by chemical and enzymic methods. 2. The results are consistent with the presence of a single carbohydrate prosthetic group, linked through its reducing end group to the peptide chain. 3. Further, all the 2-amino-2-deoxy-d-glucose units appear to be in the N-acylated form, the phenolic hydroxyl group of tyrosine is free and the ω-carboxyl group of aspartic acid is substituted. 4. The carbohydrate component has a branched-chain structure, the two non-reducing ends being terminated by a d-mannopyranosyl and a 2-acetamido-2-deoxy-d-glucopyranosyl residue respectively. 5. The terminal d-mannopyranosyl unit is probably linked through at least one other d-mannopyranosyl residue to the remainder of the carbohydrate. 相似文献
The distribution of glycosaminoglycans in disrupted glomerular fractions was studied using 35SO4-labeling in vivo and in vitro. The majority of 35S of isolated glomerular basement membrane was found in heparan sulfate after in vivo and in vitro pulses, although the absolute proportion and the degrees of N-sulfation and N-acetylation varied with the conditions of exposure. Varying amounts of chondroitin sulfate and dermatan sulfate were found in the glomerular basement membrane fraction and larger proportions of both of these glycosaminoglycans as well as of heparan sulfate were found in various glomerular fractions. Glomerular glycosaminoglycans distribution studies must take into account the experimental conditions. Basement membrane-like components of the glomerulus such as the mesangial matrix may have varying glycosaminoglycan composition which may be found in association with glomerular basement membrane fractions. 相似文献
For chemo-enzymatic synthesis of a glycosylated peptide, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was used for the synthesis of a N-acetylglucosaminyl peptide and a pseudoglycopeptide by solid-phase peptide synthesis without the requirement of protecting groups on the carbohydrate. We also performed transglycosylation of an N-glycan to the N-acetylglucosaminyl peptide using endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) to synthesize a glycopeptide containing a complex N-glycan. 相似文献
Infrared, and 1H- and 13C-NMR spectroscopy has been applied to a study of the planar interaction in apolar media between 1-substituted N4-methoxycytosine (and the corresponding 5-melhyl analogue) and 9-substituted adenines. In both chloroform and carbon tetrachloride solutions, the exocyclic N4-methoxy group of N4-methoxycytosine, and of 5-methyl N4-methoxy-cytosine (which is in the oxime form under these conditions), is so oriented that it is predominantly syn to the ring N(3), and neither compound forms planar auto-associates. In chloroform solution, both the N(3)-H and the C(2)O interact weakly with the solvent, the interaction being of the nature of non-typical hydrogen bonding The 13C-NMR chemical shift of the C(2) of N4-methoxycylosine is modified during formation of hetero-associates with 9-substituted adenine, in accordance with the C(2)O of the former being the acceptor of an adenine ammo proton. The resulting planar hetero-associate is a non-Watson-Crick type of base-pair. This was further substantiated by infrared absorption studies of the carbonyl frequency during complex formation. The results are examined in the light of the mechanism of hydroxylamine (and methoxyamine) mutagenesis. 相似文献
The Cuvierian tubules of Holothuria forskali Della Chiaje, a sea cucumber found in the Adriatic Sea, were investigated with regard to their carbohydrate moieties. From a Pronase digest of these tubules three types of carbohydrate units were isolated and characterized. 1. A high-molecular-weight glycopeptide fraction was shown to contain sulphated polyfucose, galactosamine, a uronic acid and a previously unknown neuraminic acid derivative. The sulphate was shown by i.r. analysis to be present as an O-ester. The carbohydrate unit was linked O-glycosidically to threonine and serine residues in the polypeptide chain. The hitherto unknown neuraminic acid derivative (Hf-neuraminic acid) was resistant to enzymic cleavage by neuraminidase, even after mild alkaline hydrolysis for the removal of O-acyl residues. However, the glycosidic linkage of this compound to the other part of the carbohydrate moiety was readily cleaved by mild acid hydrolysis. Its chromatographic properties distinguished Hf-neuraminic acid from other known neuraminic acid derivatives (N-acetyl-, NO-diacetyl-, NOO-triacetyl- and N-glycollyl-neuraminic acid). Further, this acidic sugar was shown to possess neuraminic acid as its basic structure. Thus, an as yet unknown substituent lends the distinct properties to Hf-neuraminic acid. 2. The carbohydrate composition of a second glycopeptide fraction consisting of a derivative of neuraminic acid, galactose, mannose and glucosamine was similar to that of the well-known carbohydrate groups of the globular glycoproteins. 3. The third fraction contained two glycopeptides containing the disaccharide, glucosylgalactose, which was shown to be linked to the hydroxyl group of hydroxylysine residues of a collagen-like protein. Approximately half of these residues were glycosylated. In addition to these glycopeptides, a small amount of a third glycopeptide that carried only a galactosyl residue was detected. The amino acid sequence of the two major compounds were found to be Gly-Ala-Hyl*-Gly-Ser and Gly-Pro-Hyl*-Gly-Asp, where Hyl* represents a glycosylated amino acid residue. 相似文献
A glycopeptide (In1) was isolated by phenol-water extraction from Cyttaria harioti Fischer, parasite of Nothofagus sps. Neutral sugars account for 89% of In1 and were characterized as glucose, mannose, and galactose. Glucosamine, identified by g.l.c., was colorimetrically estimated (5.8%). The molar ratio of Glc:Man:Gal:GlcNAc was 17:11:3:2. The linkages between the various monosaccharide residues were established through methylation analysis and periodate oxidation studies. The anomeric configurations of the various glycosyl groups were determined by chromium trioxide oxidation of the acetylated polysaccharide. The results were confirmed by 13C-n.m.r. spectroscopy. The sugar chain is N-glycosyl-linked to the peptide. Structural features of the carbohydrate moiety of glycopeptide In1 are described. 相似文献
Acid -galactosidase (EC3.2.1.23) was obtained from human liver in a pure monomeric state (Mr63 000). The carbohydrate content of the enzyme was established to be, 9% by weight; mannose,N-acetylglucosamine, galactose andN-acetylneuraminic acid were found to be the constituent monosaccharides. The carbohydrate structures of the enzyme were studied at the glycopeptide level by employing 500 MHz1H-NMR spectroscopy, carbohydrate composition analysis and methylation analysis involving GLCMS. Based upon the intensities of relevant signals in the1H-NMR spectrum, approximately 60% of the chains were found to be of theN-acetyllactosamine type, having the structure
The rest appeared to be of the oligomannoside type (Man5-6GlcNAc2Asn). The carbohydrate composition and methylation analysis results sustained these findings, although the calculation of the distribution based upon these techniques indicated a somewhat lower percentage ofN-acetyllactosamine type chains. There are approximately three oligosaccharide chains per molecule. These findings offer an explanation for the abnormal distribution of -galactosidase in tissues and cultured fibroblasts of patients with I-cell disease. 相似文献
The glycosaminoglycans of various basement membranes (human and bovine renal glomerular and tubular basement membranes as well as calf and cow anterior and posterior lens capsules) have been isolated by DEAE-cellulose chromatography after protease digestion. On the basis of composition, ion-exchange elution, electrophoretic mobility, and susceptibility to nitrous acid treatment heparan sulfate was identified as the predominant glycosaminoglycan component of each membrane. Quantitation of the heparan sulfate was achieved by a DEAE-cellulose microcolumn procedure and indicated that the amount of this component present in basement membranes spanned a wide range, extending from 0.3% of peptide weight in bovine and human tubular membranes to 6% in calf posterior lens capsule. Comparison of the heparan sulfate content of calf and cow anterior lens capsules indicated that it underwent a pronounced decrease with increasing age. Analyses of the glycosaminoglycan-peptide fractions from calf anterior and posterior lens capsules indicated hexuronic acid to xylose ratios of 29 and 37, respectively, and relatively low degrees of N-sulfation (0.2 N-sulfate, 0.6 total sulfate groups per repeating disaccharide). The composition of the lens capsule heparan sulfate was in many ways similar to that from bovine glomerular basement membrane (N. Parthasarathy and R. G. Spiro, 1981, J. Biol. Chem.256, 507–513). The present study also indicated that the heparan sulfate content of bovine glomerular basement membrane (0.8 mg/100 mg peptide) was not appreciably altered even by prolonged sonic treatment. 相似文献
Previous reports from this laboratory (1–4) described the perbenzoylation of neutral glycosphingolipids (GSL)1 with benzoyl chloride in pyridine and analysis of the perbenzoylated derivatives by high performance liquid chromatography (hplc). A disadvantage of this procedure is that N-benzoylation occurs as well as the desired O-benzoylation. This does not permit recovery of the parent GSL after mild alkaline hydrolysis due to formation of a mixture of N-acylated and N-benzoylated GSLs(1). It has also been demonstrated that the benzoylation with benzoic anhydride in pyridine does not lead to the formation of N-benzoylated products. However, the anhydride reaction is sluggish and the benzoyl chloride method has been the preferred procedure.Gupta et al. (5) used N,N-dimethyl-4 amino pyridine (DMAP) as a catalyst in the acylation of phospholipids by the anhydrides of fatty acids. F. B. Jungalwala (private communication) has shown that this catalyst greatly accelerates the reaction of benzoic anhydride with sulfatides.In this communication we report the preparation and hplc analysis of per-O-benzoyl derivatives of GSLs by reaction with benzoic acid anhydride in the presence of DMAP as a catalyst. Reaction with these reagents avoids amide acylation, forms single products with satisfactory chromatographic properties and parent GSLs can be regenerated by mild alkaline hydrolysis. 相似文献
The 500-MHz1H-NMR characteristics of theN-linked carbohydrate chain Man1-6[Xyl1-2]Man1-4GlcNAc1-4[Fuc1-3]GlcNAc1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The1H-NMR structural-reporter-groups of the (1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide. 相似文献
The complexes (Tpms)VCl2(DMF) (1), and (Tpms)VOCl(DMF) (2), have been synthesized and characterized. Complex 2 has also been structurally characterized via X-ray diffractometry. The vanadium(IV) center possesses a square pyramidal/distorted octahedral geometry with a facially coordinating Tpms− ligand in a κ3-N,N,O coordination mode. The complex is the first structurally characterized example of a vanadium(IV) complex with Tpms−. Complex 2 shows catalytic activity towards oxidation of 3,5-di-tert-butyl catechol and also exhibits phosphatase inhibition characteristics on alkaline phosphatase. Tpms− = trispyrazolylmethanesulfonate; DMF = N,N-dimethylformamide. 相似文献
Autoantibodies to components of chromatin, which include double-stranded DNA (dsDNA), histones and nucleosomes, are central in the pathogenesis of lupus nephritis. How anti-chromatin autoantibodies exert their nephritogenic activity, however, is controversial. One model assumes that autoantibodies initiate inflammation when they cross-react with intrinsic glomerular structures such as components of membranes, matrices or exposed nonchromatin ligands released from cells. Another model suggests glomerular deposition of autoantibodies in complex with chromatin, thereby inducing classic immune complex–mediated tissue damage. Recent data suggest acquired error of renal chromatin degradation due to the loss of renal DNaseI enzyme activity is an important contributing factor to the development of lupus nephritis in lupus-prone (NZBxNZW)F1 mice and in patients with lupus nephritis. Down-regulation of DNaseI expression results in reduced chromatin fragmentation and in deposition of extracellular chromatin–IgG complexes in glomerular basement membranes in individuals who produce IgG anti-chromatin autoantibodies. The main focus of the present review is to discuss whether exposed chromatin fragments in glomeruli are targeted by potentially nephritogenic anti-dsDNA autoantibodies or if the nephritogenic activity of these autoantibodies is explained by cross-reaction with intrinsic glomerular constituents or if both models coexist in diseased kidneys. In addition, the role of silencing of the renal DNaseI gene and the biological consequences of reduced chromatin fragmentation in nephritic kidneys are discussed. 相似文献
We have carried out detailed structural studies of the glycopeptides of glycoprotein gD of herpes simplex virus types 1 and 2. We first examined and compared the number of N-asparagine-linked oligosaccharides present in each glycoprotein. We found that treatment of either pgD-1 or pgD-2 with endo-β-N-acetylglucosaminidase H (Endo H) generated three polypeptides which migrated more rapidly than pgD on gradient sodium dodecyl sulfate-polyacrylamide gels. Two of the faster-migrating polypeptides were labeled with [3H]mannose, suggesting that both pgD-1 and pgD-2 contained three N-asparagine-linked oligosaccharides. Second, we characterized the [3H]mannose-labeled tryptic peptides of pgD-1 and pgD-2. We found that both glycoproteins contained three tryptic glycopeptides, termed glycopeptides 1, 2, and 3. Gel filtration studies indicated that the molecular weights of these three peptides were approximately 10,000, 3,900, and 1,800, respectively, for both pgD-1 and pgD-2. Three methods were employed to determine the size of the attached oligosaccharides. First, the [3H]mannose-labeled glycopeptides were treated with Endo H, and the released oligosaccharide was chromatographed on Bio-Gel P6. The size of this molecule was estimated to be approximately 1,200 daltons. Second, Endo H treatment of [35S]methionine-labeled glycopeptide 2 reduced the molecular size of this peptide from approximately 3,900 to approximately 2,400 daltons. Third, glycopeptide 2 isolated from the gD-like molecule formed in the presence of tunicamycin was approximately 2,200 daltons. From these experiments, the size of each N-asparagine-linked oligosaccharide was estimated to be approximately 1,400 to 1,600 daltons. Our experiments indicated that glycopeptides 2 and 3 each contained one N-asparagine-linked oligosaccharide chain. Although glycopeptide 1 was large enough to accommodate more than one oligosaccharide chain, the experiments with Endo H treatment of the glycoprotein indicated that there were only three N-asparagine-linked oligosaccharides present in pgD-1 and pgD-2. Further studies of the tryptic glycopeptides by reverse-phase high-performance liquid chromatography indicated that all of the glycopeptides were hydrophobic in nature. In the case of glycopeptide 2, we observed that when the carbohydrate was not present, the hydrophobicity of the peptide increased. The properties of the tryptic glycopeptides of pgD-1 were compared with the properties predicted from the deduced amino acid sequence of gD-1. The size and amino acid composition compared favorably for glycopeptides 1 and 2. Glycopeptide 3 appeared to be somewhat smaller than would be predicted from the deduced sequence of gD-1. It appears that all three potential glycosylation sites predicted by the amino acid sequence are utilized in gD-1 and that a similar number of glycosylation sites are present in gD-2. 相似文献
The effects of treatments of the glycoprotein ribonuclease-B, the proteins ribonuclease-A and myoglobin, and the glyco-amino acid GlcNAc(1-N) Asn with alkalil alkaline sodium borohydride, and aqueous sodium borohydride were systematically studied as a function of the concentration of the reagents, the temperature, and the length of the treatment. High-field1H-NMR spectroscopy, chromatographic methods and amino-acid analysis were used to characterize products of the treatments of the various compounds. Our results indicate that mild alkaline borohydride treatment, as well as aqueous borohydride treatment alone, is capable of extensively degrading polypeptides and of partially releasing theN-linked glycans from ribonuclease-B. Initially, glycopeptides are produced, the peptide portion of which consists of several amino acids, which are further hydrolyzed to yield a mixture of glyco-asparagines and oligosaccharide-alditols in the ratio of 4:1. Strong alkaline borohydride treatment of ribonuclease-B is capable of completely releasing theN-linked carbohydrates as oligosaccharide-alditols.Abbreviation RNase
ribonuclease 相似文献
Bovine thyroid glands are known to contain a complex array of gangliosides. One of the predominant gangliosides was isolated and analyzed by gas-liquid chromatography and mass spectrometry. The carbohydrate composition was fucose, N-acetylneuraminic acid, galactose, N-acetylgalactosamine, and glucose in molar ratios of 1:1:2:1:1. The structure of the ganglioside was identified as: 相似文献