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1.
J Lewandowski 《Analytical biochemistry》1973,54(2):325-332
A simple method of determinations of three hexosamines is presented. The solutions of hexosamines heated in 1 n NaOH at 60°C for 60 min, absorb ultraviolet light with the maximum at 302 nm. A linear relationship exists between the concentration of hexosamine up to 100 μg and the absorbance at 302 nm. N-acetylated hexosamines, having been deacetylated, may be determined in this way. The characteristic absorption curve ranging from 225 to 360 nm with the maximum at 302 nm may be utilized as a qualitative criterion for identification of hexosamines and N-acetylated hexosamines after their deacetylation. 相似文献
2.
Hexosamine biosynthesis and incorporation into polysaccharides glyco-proteins have been studied in four species of fungi—Aspergillus niger, Penicillium citrinum, Cladosporium cladosporoides, and Fusarium moniliforme. Hydrolytic, recovery, and analytical methods are described for the estimation of hexosamine accumulation in fungal growth. Optimum yields of hexosamine from matrix are achieved by using hydrolysis with strong acid (8M hydrochloric acid) over a period of 2–3 h. For all fungal-growth studies, hexosamine was quantitated, after hydrolysis, by an automated, amino acid analyzer programmed for the separation of amino sugars. Methods were also developed, using gas-liquid chromatography (nitrogen-selective alkaline flame-ionization detector and trimethylsilyl derivatives of hexosamines), and the modified Morgan-Elson reaction of N-acetylated hexosamines. Both the amino acid analyzer and gas-chromatographic method quantify nmol amounts of hexosamine per mg of dry-weight sample. In all phases of the growth cycle, a linear correlation was found for the four fungi between the amount of hexosamine per unit time, age of culture, and quantity of mycelial biomass in liquid medium. With solid corn-meal as a fungal growth-medium, samples also demonstrated a linear correlation, but only between hexosamine biosynthesis and age of culture, as biomass was not determinable. Autolysis of hexosamine occurs to only a limited extent during late stationary-phase in liquid medium and on corn. 相似文献
3.
A 96-well plate method was developed for analysis of total hexosamine content in biological samples. Four hexosamine monomer derivatives—glucosamine hydrochloride, glucosamine sulfate, galactosamine hydrochloride, and mannosamine hydrochloride—were examined for the linearity of their spectra in the concentration range specified in the assay. The hexosamine concentration analysis range was linear from 0.1 to 1 mM. The quantification of hexosamines from chitin and chitosan upon acid hydrolysis was also tested. Accurate quantification of glucosamine content in chitin and chitosan with different molecular sizes and degrees of acetylation was demonstrated using the new method. 相似文献
4.
S Ambrosio J R De Oliveira R Bartrons 《Journal of biochemical and biophysical methods》1992,25(4):237-244
Galactosamine is quickly metabolized to galactosamine 1-phosphate in rats treated with this compound. An HPLC method to quantify hexosamine phosphates in biological samples is described, modified from the o-phthaldialdehyde amino acid analysis procedure. o-Phthaldialdehyde derivatives of hexosamines and hexosamine-phosphates can be eluted from a reverse-phase column at different retention times, with a total analysis time of 30 min and without overlapping with free amino acids at physiological concentrations. The standard curves are linear between 1 and 40 nmol. This simple method is more selective and sensitive than previous enzymatic analyses of hexosamine phosphorylation. 相似文献
5.
Suguru Suzuki Sakaru Suzuki Nobuto Nakamura Takeo Koizumi 《Biochimica et Biophysica Acta (BBA)/General Subjects》1976,428(1):166-181
The glycosaminoglycan of rat liver can be separated into five distinct fractions; a hyaluronic acid franction, a heparan sulfate fraction with a molar ratio of sulfate to hexosamine (S/HexN) around 0.7, a heparan sulfate fraction with a S/HexN ratio around 1.4, a dermatan sulfate fraction with a S/HexN ratio near unity, and a dermatan sulfate fraction with a S/HexN ratio around 1.3.Enzymatic analysis of the two dermatan sulfate fractions indicates that they differ significantly in that the high sulfated fraction contains relatively more N-acetylgalactosamine 4,6-bissulfate units (about 26% of the total hexosamine). In experimental injury produced by carbon tetrachloride, the low sulfated fraction increases as much as 9-fold on a dry weight basis, bearing no linear relationship to the amount of the high sulfated fraction which increases only 2-fold. A significant shift is also observed in the levels of the two heparan sulfate fractions. In this case, however, the high sulfated fraction shows a much more pronounced increase than does the low sulfated fraction. On the basis of these observations, it is suggested that for each of the dermatan sulfate and heparan sulfate classes are at least two pools, distinguished by sulfation degree and perhaps by turnover rate and physiological function. 相似文献
6.
Podyma-Inoue KA Yokote H Sakaguchi K Ikuta M Yanagishita M 《The Journal of biological chemistry》2002,277(36):32459-32465
Cell surface heparan sulfate proteoglycans undergo unique intracellular degradation pathways after they are endocytosed from the cell surface. Heparanase, an endo-beta-glucuronidase capable of cleaving heparan sulfate, has been demonstrated to contribute to the physiological degradation of heparan sulfate proteoglycans and therefore regulation of their biological functions. A rat parathyroid cell line was found to produce heparanase with an optimal activity at neutral and slightly acidic conditions suggesting that the enzyme participates in heparan sulfate proteoglycan metabolism in extralysosomal compartments. To elucidate the detailed properties of the purified enzyme, the substrate specificity against naturally occurring heparan sulfates and chemically modified heparins was studied. Cleavage sites of rat heparanase were present in heparan sulfate chains obtained from a variety of animal organs, but their occurrence was infrequent (average, 1-2 sites per chain) requiring recognition of both undersulfated and sulfated regions of heparan sulfate. On the other hand intact and chemically modified heparins were not cleaved by heparanase. The carbohydrate structure of the newly generated reducing end region of heparan sulfate cleaved by the enzyme was determined, and it represented relatively undersulfated structures. O-Sulfation of heparan sulfate chains also played important roles in substrate recognition, implying that rat parathyroid heparanase acts near the boundary of highly sulfated and undersulfated domains of heparan sulfate proteoglycans. Further elucidation of the roles of heparanase in normal physiological processes would provide an important tool for analyzing the regulation of heparan sulfate-dependent cell functions. 相似文献
7.
Venous and aortic porcine endothelial cells cultured under standardized conditions synthesize heparan sulfate chains which differ in charge 总被引:2,自引:0,他引:2
The identification of a specific required carbohydrate structure for the antithrombin III binding site on heparin suggests that there may be specific structures in glycosaminoglycan chains which are necessary for other vascular functions of these carbohydrates. Determining that such differences exist requires a mechanism to isolate heparan sulfates from endothelial cells of specific vascular beds. The present report indicates that cultured venous and aortic endothelial cells synthesize heparan sulfate chains differing in charge density. There are two important conclusions from this work. (i) Endothelial cells from different blood vessels (i.e., vena cava and thoracic aorta) synthesize heparan sulfates which differ in negative charge and sulfation pattern. Specifically, aortic endothelial heparan sulfates have a higher negative charge than venous heparan sulfates. Differences are also observed in the nitrous acid degradation products of the heparan sulfates. (ii) Endothelial cells in culture retain the ability to synthesize different heparan sulfates in vitro after months of subculture under defined conditions. These results indicate that it is feasible to characterize heparan sulfates using cultured endothelial cells from a variety of vascular beds. 相似文献
8.
Suzuki K Yamamoto K Kariya Y Maeda H Ishimaru T Miyaura S Fujii M Yusa A Joo EJ Kimata K Kannagi R Kim YS Kyogashima M 《Glycoconjugate journal》2008,25(8):703-712
Five monoclonal antibodies AS17, 22, 25, 38 and 48, a single monoclonal antibody ACH55, and three monoclonal antibodies NAH33,
43, 46, that recognize acharan sulfate (IdoA2S-GlcNAc)n, acharan (IdoA-GlcNAc)n and N-acetyl-heparosan (GlcA-GlcNAc)n, respectively, were generated by immunization of mice with keyhole limpet hemocyanin-conjugated
polysaccharides. Specificity tests were performed using a panel of biotinylated GAGs that included chemically modified heparins.
Each antibody bound avidly to the immunized polysaccharide, but did not bind to chondroitin sulfates, keratan sulfate, chondroitin
nor hyaluronic acid. AS antibodies did not bind to heparan sulfate or heparin, but bound to 6-O-desulfated, N-desulfated and re-N-acetylated heparin to varying degrees. ACH55 bound to tri-desulfated and re-N-acetylated heparin but hardly bound to other modified heparins. NAH antibodies did not bind to heparin and modified heparins
but bound to heparan sulfate to varying degrees. NAH43 and NAH46 also bound to partially N-de-acetylated N-acetyl-heparosan. Immunohistochemical analysis in rat cerebella was performed with the antibodies. While NAH46 stained endothelia,
where heparan sulfate is typically present, neither ACH55 nor AS25 stained endothelia. On the contrary ACH55 and AS25 stained
the molecular layer of the rat cerebella. Furthermore, ACH55 specifically stained Purkinje cells. These results suggest that
there is unordinary expression of IdoA2S-GlcNAc and IdoA-GlcNAc in specific parts of the nervous system.
Suzuki and Yamamoto contributed equally to this study. 相似文献
9.
《Journal of molecular recognition : JMR》2017,30(3)
Heparan sulfates are complex polysaccharides belonging to the family of glycosaminoglycans that participate to the regulation of cell behavior and tissue homeostasis. The biological activities conferred to heparan sulfates are largely dependent on the content and positioning of the sulfate groups along their saccharidic units. At present, identification of particular sulfation patterns in biologically relevant heparan sulfate sequences remains challenging. Although several approaches for structure analysis exist, the complexity of heparan sulfates makes new and original approaches still required. Here, we used molecular imprinting technologies to prepare a library of polyethylene glycol acrylate functionalized hydrogels with the aim to investigate their applicability as specific recognizing systems for fondaparinux, a synthetic pentasaccharide analog to the antithrombin binding site of heparin. Adequate choice of the hydrogel composition and controlling rebinding conditions were important determinants for improving the sulfated oligosaccharide recognition specificity and selectivity. Our results suggest that molecular imprinting approaches could be a possibility for the specific recognition of biologically active sequences in heparan sulfates. 相似文献
10.
Heparan sulfate: decoding a dynamic multifunctional cell regulator 总被引:12,自引:0,他引:12
The heparan sulfates are a family of cell-surface and matrix polysaccharides with an incredible degree of structural diversity that are distributed widely in virtually all metazoan organisms. Recent genetic, biochemical and cell-biological studies have led to increased understanding of the biosynthetic mechanisms that produce these complex molecules, as well as their functional versatility in regulating protein activities. The dynamic expression of heparan sulfates with differing sugar sequences suggests a new concept in which the repertoire of sequences produced by a particular cell or tissue is designated its 'heparanome'. This review discusses recent developments and surveys emerging experimental strategies that hold promise for revealing the functional specificity and mechanisms of action of heparan sulfates as multifunctional cell regulators. 相似文献
11.
Sophie M. Thompson David G. Fernig Edwin C. Jesudason Paul D. Losty Els M. A. van de Westerlo Toin H. van Kuppevelt Jeremy E. Turnbull 《The Journal of biological chemistry》2009,284(51):35621-35631
Heparan sulfate (HS) binds and modulates the transport and activity of a large repertoire of regulatory proteins. The HS phage display antibodies are powerful tools for the analysis of native HS structure in situ; however, their epitopes are not well defined. Analysis of the binding specificities of a set of HS antibodies by competitive binding assays with well defined chemically modified heparins demonstrates that O-sulfates are essential for binding; however, increasing sulfation does not necessarily correlate with increased antibody reactivity. IC50 values for competition with double modified heparins were not predictable from IC50 values with corresponding singly modified heparins. Binding assays and immunohistochemistry revealed that individual antibodies recognize distinct epitopes and that these are not single linear sequences but families of structurally similar motifs in which subtle variations in sulfation and conformation modify the affinity of interaction. Modeling of the antibodies demonstrates that they possess highly basic CDR3 and surrounding surfaces, presenting a number of possible orientations for HS binding. Unexpectedly, there are significant differences between the existence of epitopes in tissue sections and observed in vitro in dot blotted tissue extracts, demonstrating that in vitro specificity does not necessarily correlate with specificity in situ/vivo. The epitopes are therefore more complex than previously considered. Overall, these data have significance for structure-activity relationships of HS, because the model of one antibody recognizing multiple HS structures and the influence of other in situ HS-binding proteins on epitope availability are likely to reflect the selectivity of many HS-protein interactions in vivo. 相似文献
12.
During the hydrolysis of an exopolysaccharide (EPS) produced by Citrobacter sp., the maximum liberation of hexosamine was obtained with 6 m HCl at 115 °C in an autoclave for 1 h. The glycosidic bond energy and degree of acetylation of the hexosamine in EPS were approximately 77 kJ mol–1 and 61%, respectively. Thermal destruction of the hexosamines and the effect of salt on the hexosamine determination could be minimized under the optimized hydrolytic conditions. Using a modified Elson–Morgan method, maximum total hexosamine concentration was determined to be 3.2 g l–1 (29% of crude EPS) after 96 h of fed-batch culture.Revisions requested 18 August 2004; Revisions received 2 November 2004 相似文献
13.
Isolation and structural studies of heparan sulfates and chondroitin sulfates from three species of molluscs 总被引:5,自引:0,他引:5
H B Nader T M Ferreira J F Paiva M G Medeiros S M Jer?nimo V M Paiva C P Dietrich 《The Journal of biological chemistry》1984,259(3):1431-1435
The isolation and some structural features of heparan sulfates and chondroitin sulfates from three species of molluscs (Pomacea sp., Tagelus gibbus, and Anomalocardia brasiliana) are reported. It is shown that heparan sulfates with structural similarities to the ones found in mammals are present in the three molluscs analyzed. All the heparan sulfates were degraded by heparitinases I and II to four distinct unsaturated disaccharides with the same properties as the ones present in heparan sulfates of mammalian origin. This suggests that these four disaccharide units are maintained through the evolution. Furthermore, the proportion of these units varied in the heparan sulfates according to the species of origin. The chondroitin sulfates, on the other hand, exhibit different structural features according to the species of origin. For instance, by the action of chondroitinase AC, 4- and nonsulfated disaccharides are produced from Pomacea chondroitin, whereas 4- and 6-sulfated disaccharides are formed from Tagelus and Anomalocardia. The possible role of these compounds in cell recognition and/or adhesiveness is discussed in view of the present findings. 相似文献
14.
An unusual heparan sulfate was isolated from lobsters (Homarus americanus). The polysaccharide has a composition and properties intermediate to heparin and the more common heparan sulfates. The sulfate and D-glucuronic acid content is high, while anticoagulant activity is low. The major repeating unit appears to consist of D-glucuronic acid and D-glucosamine N-O-disulfate. Some N-acetyl groups are present, the content of L-iduronic acid O-sulfate is low, and monosulfated or nonsulfated disaccharide-repeating units (very common in heparan sulfates) appear to be very rare. The data obtained again emphasize the heterogeneity of heparan sulfates and the need for adequate characterization when dealing with unusual or unexplored sources. 相似文献
15.
The glycosaminoglycan of rat liver can be separated into five distinct fractions; a hyaluronic acid fraction, a heparan sulfate fraction with a molar ratio of sulfate to hexosamine (S/HexN) around 0.7, a heparan sulfate fraction with a S/HexN ratio around 1.4, a dermatan sulfate fraction with a S/HexN ratio near unity, and a dermatan sulfate fraction with a S/HexN ratio around 1.3. Enzymatic analysis of the two dermatan sulfate fractions indicates that they differ significantly in that the high sulfated fraction contains relatively more N-acetylgalactosamine 4,6-bissulfate units (about 26% of the total hexosamine). In experimental injury produced by carbon tetrachloride, the low sulfated fraction increases as much as 9-fold on a dry weight basis, bearing no linear relationship to the amount of the high sulfated fraction which increases only 2-fold. A significant shift is also observed in the levels of the two heparan sulfate fractions. In this case, however, the high sulfated fraction shows a much more pronounced increase than does the low sulfated fraction. On the basis of these observations, it is suggested that for each of the dermatan sulfate and heparan sulfate classes there are at least two pools, distinguished by sulfation degree and perhaps by turnover rate and physiological function. 相似文献
16.
Maria E. Silva Carl P. Dietrich Helena B. Nader 《Biochimica et Biophysica Acta (BBA)/General Subjects》1976,437(1):129-141
The analyses of the products formed from heparitin sulfates by the action of two heparitinases and a heparinase from Flavobacterium heparinum is reported. Heparitin sulfates A and B are degraded by heparitinase I yielding two disaccharides, one of them composed of N-acetylglucosamine and an unsaturated uronic, joined by α(1 → 4) linkage, and the other, with the same composition but with an O-sulfate at the hexosamine moiety. A third disaccharide is also formed from heparitin sulfate B, by the action of the same enzyme, composed of glucosamine N-sulfate and an unsaturated uronic acid joined probably by α(1 → 4) linkage. Besides these three disaccharides, heparitin sulfate B yields, by the action of heparitinase I, an oligosaccharide (with an average molecular weight of 6000) which is completely degraded by the heparitinase II yielding a disaccharide composed of glucosamine 2,6-disulfate and unsaturated uronic acid. All the disaccharides are further degraded by α-glycuronidase from Flavobacterium heparinum yielding the respective monosaccharides. Based on these and other analyses the possible structures of the heparitin sulfates are proposed. 相似文献
17.
Richard C Roghani M Moscatelli D 《Biochemical and biophysical research communications》2000,276(2):399-405
In the presence of FGF-2, cells in suspension expressing FGF receptor-1 will attach to monolayers of cells expressing heparan sulfates. This attachment provides physical evidence for the formation of a trimolecular complex between FGF-2, heparan sulfate, and FGF receptors. We have used this system to determine if receptor isoforms containing or lacking the first of three immunoglobulin-like domains are equally able to form complexes with FGF-2 and heparan sulfates. In the presence of FGF-2, cells expressing either isoform of the receptor were able to attach to monolayers of CHO cells expressing heparan sulfates. No attachment was observed in the absence of FGF-2 or if heparin was included in the incubation medium. Attachment of cells expressing the two receptor isoforms occurred at similar concentrations of FGF-2, and similar concentrations of heparin were required to disrupt the interactions. Thus, there appeared to be little difference between these receptor isoforms in their ability to form trimolecular complexes with FGF-2 and cell-associated heparan sulfates. We also found that, in the presence of FGF-2, cells expressing FGF receptor-1 are able to form complexes with both extracellular matrix and cell-surface heparan sulfates. 相似文献
18.
Synthesis of sulfated glycosaminoglycans by embryonic corneal epithelium 总被引:11,自引:0,他引:11
The primary corneal stroma is produced by the overlying epithelium. The endothelium appears between 4 and 5 days, fibroblasts at 6 days, and at 12 days the epithelium stratifies. We investigated the synthesis of glycosaminoglycan (GAG) by the epithelium during this developmentally significant period. The sulfated GAG synthesized by isolated 4–6-day-old corneal epithelia during the first 24 hr in vitro are entirely accountable for as chondroitin sulfates and heparan sulfates. Nearly 50% of the total sulfated GAG synthesized by epithelia on Millipore filters is lost to the medium, but only 30–40% is lost when frozen killed lens capsule or stroma is the substratum. Retention of isotope by the tissue is correlated with visible matrix polymerization. The relative amount of heparan sulfate synthesized by the developing epithelium 24 hr in vitro decreases from about 50% of the total sulfated GAG for 4-day-old epithelium to 12% for 12-day-old epithelium. A similar decrease in heparan sulfate synthesis occurs with time in culture. The relative amount of GAG identified as chondroitin sulfate and heparan sulfate is the same when 3H-glucosamine is used to label GAG as when 35SO4 is used. We conclude that the corneal epithelium produces only sulfated polysaccharides. Since hyaluronate is synthesized by whole 5-day-old corneas, it must be the product of the endothelium. 相似文献
19.
Anchorage of collagen-tailed acetylcholinesterase to the extracellular matrix is mediated by heparan sulfate proteoglycans 总被引:8,自引:3,他引:5
Heparan sulfate and heparin, two sulfated glycosaminoglycans (GAGs), extracted collagen-tailed acetylcholinesterase (AChE) from the extracellular matrix (ECM) of the electric organ of Discopyge tschudii. The effect of heparan sulfate and heparin was abolished by protamine; other GAGs could not extract the esterase. The solubilization of the asymmetric AChE apparently occurs through the formation of a soluble AChE-GAG complex of 30S. Heparitinase treatment but not chondroitinase ABC treatment of the ECM released asymmetric AChE forms. This provides direct evidence for the vivo interaction between asymmetric AChE and heparan sulfate residues of the ECM. Biochemical analysis of the electric organ ECM showed that sulfated GAGs bound to proteoglycans account for 5% of the total basal lamina. Approximately 20% of the total GAGs were susceptible to heparitinase or nitrous acid oxidation which degrades specifically heparan sulfates, and approximately 80% were susceptible to digestion with chondroitinase ABC, which degrades chondroitin-4 and -6 sulfates and dermatan sulfate. Our experiments provide evidence that asymmetric AChE and carbohydrate components of proteoglycans are associated in the ECM; they also indicate that a heparan sulfate proteoglycan is involved in the anchorage of the collagen-tailed AChE to the synaptic basal lamina. 相似文献
20.
Kayitmazer AB Quinn B Kimura K Ryan GL Tate AJ Pink DA Dubin PL 《Biomacromolecules》2010,11(12):3325-3331
Long-range electrostatic interactions are generally assigned a subordinate role in the high-affinity binding of proteins by glycosaminoglycans, the most highly charged biopolyelectrolytes. The discovery of high and low sulfation domains in heparan sulfates, however, suggests selectivity via complementarity of their linear sulfation patterns with protein charge patterns. We examined how charge sequences in anionic/nonionic copolymers affect their binding to a protein with prominent charge anisotropy. Experiments and united-atom Monte Carlo simulations, together with Delphi electrostatic modeling for the protein, confirm strongest binding when polyanion sequences allow for optimization of repulsive and attractive electrostatics. Simulations also importantly identified retention of considerable polyion conformational freedom, even for strong binding. The selective affinity for heparins of high and low charge density found for this protein is consistent with nonspecific binding to distinctly different protein charge domains. These findings suggest a more nuanced view of specificity than previously proposed for heparinoid-binding proteins. 相似文献