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PubMed1981

Hormonal effects on the sulfation of sulfated glycoprotein in a particulate fraction of the endometrium of rabbit uterus.

A particulate fraction was separated from endometrial scrapings of the uterus of hormone- or sham-treated ovariectomized rabbit. The effects of estrogen and progesterone on the incorporation of [35S]sulfate from 3'-[35S]phosphoadenosine 5'-phosphosulfate (PAPS) into endogenous acceptors in the particulate fraction were investigated. Estrogen increased the incorporation of [35S]sulfate, but progesterone suppressed this effect. The results of DEAE-Sephadex A-25 (Cl-form) column chromatography of the pronase digest of the 35S-labeled substances indicated that the fraction eluted with 0.9 M NaCl (0.9 Fr) was most sensitive to the hormones. The major component in 0.9 Fr was resistant to crude heparinase, whereas the minor component was susceptible to this enzyme. The present observation, together with previous findings, suggested that the former was sulfated glycopeptide and the latter heparan sulfate. The results of the present study indicated that PAPS can serve as the direct "activated" sulfate donor in the enzymatic sulfation of sulfated glycoprotein in the particulate fraction, that the sulfation is greatly stimulated by pre-treatment of the rabbit with estrogen, and that the estrogen effect is suppressed by progesterone.

Animals↗
PubMed1980

Regulation of Sulfate Assimilation in Tobacco Cells: EFFECT OF NITROGEN AND SULFUR NUTRITION ON SULFATE PERMEASE AND O-ACETYLSERINE SULFHYDRYLASE.

The effect of nitrogen and sulfur nutrition on sulfate permease and O-acetylserine sulfhydrylase was studied in tobacco cells.Sulfate transport rates increased 10-fold in cells transferred to sulfur-deficient B-5 medium. The addition of either sulfate or l-cysteine reduced transport 95 and 80%, respectively. The pools of sulfate, cysteine, glutathione, and methionine declined in sulfur-starved cells. The addition of either sulfate or l-cysteine increased the pools of sulfur-containing compounds, but major quantitative differences were measured. Nitrogen-starved cells had low transport rates which were not increased by addition of nitrate/ammonia. The pools of sulfate, cysteine, and methionine were high in nitrogen-starved cells and remained high upon addition of a nitrogen source. The results show that sulfate transport is regulated by the intracellular sulfate pool.O-Acetylserine sulfhydrylase was not affected by sulfur nutrition. The extractable activity was high in B-5-grown cells, sulfur-deficient cells, and cells to which either sulfate or l-cysteine had been added. In contrast, the enzyme declined in cells transferred to nitrogen-deficient medium and the amount of enzyme/g fresh weight increased 10-fold when nitrate/ammonia was added. The addition of nitrate/ammonia had no effect on the cysteine or methionine pools but increased the total amino acid pool. The amount of O-acetylserine was positively correlated with extractable enzyme activity. This enzyme is positively regulated by an effector (possibly O-acetylserine) which is high under conditions of net nitrate assimilation.

Journal Article↗
PubMed1975

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. I. Characterization of different pools of sulfated glycosaminoglycans.

"Fibroblast-like" cells from the intimal layer of bovine aorta were grown in culture. The formation, composition, molecular weight and turnover rate of different pools of glycosaminoglycans were investigated in cultures incubated in the presence [35S]sulfate or [14C]glucosamine. The newly synthesized glycosaminoglycans are distributed into an extracellular pool (37 - 58%), a cell-membrane associated or pericellular pool (23 - 33%), and an intracellular pool (19 - 30%), each pool exhibiting a characteristic distribution pattern of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronate. The distribution pattern of the extracellular glycosaminoglycans resembles closely that found in bovine aorta. A small subfraction of the pericellular pool - tentatively named "undercellular" pool--has been characterized by its high heparan sulfate content. The intracellular and pericellular [35S]glycosaminoglycan pools reach a constant radioactivity after 8-12 h and 24 h, respectively, whereas the extracellular [35S]glycosaminoglycans are secreted into the medium at a linear rate over a period of at least 6 days. The intracellular glycosaminoglycans are mainly in the process of degradation, as indicated by their low molecular weight and by their half-life of 7 h, but intracellular dermatan sulfate is degraded more rapidly (half-life 4-5 h) than intracellular chondroitin sulfate and heparan sulfate (half-life 7-8 h). Glycosaminoglycans leave the pericellular pool with a half-life of 12-14 h by 2 different routes: about 60% disappear as macromolecules into the culture medium, and the remainder is pinocytosed and degraded to a large extent. Extracellular and at least a part of the pericellular glycosaminoglycans are proteoglycans. Even under dissociative conditions (4M guanidinium chloride) their hydrodynamic volume is sufficient for partial exclusion from Sepharose 4B gel. The existence of topographically distinct glycosaminoglycan pools with varying metabolic characteristics and differing accessibility for degradation requiresa reconsideration and a more reserved interpretation of results concerning the turnover rates of glycosaminoglycans as determined in arterial tissue.

Animals↗
PubMed1988

Sulfated N-linked oligosaccharides in mammalian cells. III. Characterization of a pancreatic carcinoma cell surface glycoprotein with N- and O-sulfate esters on asparagine-linked glycans.

In the preceding two papers, we described two new classes of sulfated N-linked oligosaccharides isolated from total cellular 35SO4-labeled macromolecules of different mammalian cell lines. The first class carries various combinations of sialic acids and 6-O-sulfate esters on typical complex-type chains, while the second carries heparin and heparan-like sequences. In this study, we have characterized a sulfophosphoglycoprotein of 140 kDa from FG-Met-2 pancreatic cancer cells whose oligosaccharides share some properties of both these classes. The molecule was localized to the cell surface by electron microscopy using a monoclonal antibody (S3-53) and by cell surface 125I-labeling. Metabolic labeling of the cells with radioactive glucosamine, methionine, inorganic sulfate, or phosphate all demonstrated a single 140-kDa molecule. Pulse-chase analysis and tunicamycin treatment indicated the glycosylation of a putative primary translation product of 110 kDa via an intermediate (120 kDa) to the mature form (140 kDa). Digestion with peptide:N-glycosidase F (PNGaseF) indicated a minimum of four N-linked glycosylation sites. PNGaseF released more than 90% of the [6-3H]GlcNH2 label and 40-70% of 35SO4 label from the immunoprecipitated 140-kDa molecule. The isolated oligosaccharides were characterized as described in the preceding two papers. The majority of [6-3H]GlcNH2-labeled molecules were susceptible to neuraminidase. More than 50% of the 35SO4 label was associated with only 5-10% of the 3H-labeled chains. Some of the sulfated chains were partly sialylated molecules with four to five negative charges. Treatment with nitrous acid released about 25% of the 35SO4 label as free sulfate, together with 6% of the [6-3H]GlcNH2 label, indicating the presence of N-sulfated glucosamine residues. Some of these oligosaccharides were degraded by heparinase and heparitinase. Therefore, while they are not as highly charged as typical heparin or heparan chains, they must share structural features that permit recognition by the enzymes. Thus, this 140-kDa glycoprotein contains at least four asparagine-linked chains substituted with a heterogeneous mixture of sulfated sequences. The heterogeneity of these molecules is as extensive as that described for whole-cell sulfated N-linked oligosaccharides in the preceding two papers.

Asparagine↗
PubMed1981

Synthesis and biological activity of vitamin D3 3 beta-sulfate. Role of vitamin D3 sulfates in calcium homeostasis.

To determine the biological activity of vitamin D sulfates, we synthesized vitamin D3 3 beta-sulfate and tested its biological activity in vitamin D-deficient hypocalcemic rats. When vitamin D3 sulfate was administered as a single oral dose of 208,000 or 416,000 pmol (100 micrograms or 200 micrograms), it increased active calcium transport in the duodenum and was also able to mobilize calcium from bone and soft tissue. Dose levels below this failed to elicit a response. Vitamin D3 itself was active at doses as low as 260 pmol when administered in this manner. In order to test the biological activity of vitamin D3 sulfate in various doses when administered chronically, we tested the biological activity of vitamin D3 sulfate after 5 days or oral dosing: vitamin D3 sulfate was active at doses of 52,000 pmol/day (25 microgram), whereas vitamin D3 was active at doses of 65 to 260 pmol/day over a period of 5 days. When administered as a single intravenous dose, vitamin D3 sulfate exhibited no biological activity in doses as high as 52,000 pmol. Vitamin D3, however, was active at a dose of as low as 65 pmol. We conclude that vitamin D3 sulfate, a metabolite of vitamin D3 of heretofore unknown biological activity, is considerably less active than vitamin d3 itself.

Animals↗
PubMed2004

Use of 3H-glucosamine and 35S-sulfate with cultured human chondrocytes to determine the effect of glucosamine concentration on formation of chondroitin sulfate.

OBJECTIVE: To determine whether addition of glucosamine will stimulate synthesis of chondroitin sulfate by cultures of human chondrocytes, and to compare the relative contribution of endogenous glucosamine to exogenous glucosamine in forming chondroitin sulfate. METHODS: Cultured human chondrocytes were incubated with (35)S-sulfate and various amounts of glucosamine to determine whether any incremental formation of chondroitin (35)S-sulfate occurred. Similarly, chondrocytes incubated with variable concentrations of (3)H-glucosamine were examined to determine how much the incorporation into (3)H-chondroitin sulfate was diluted by provision of endogenous glucosamine that was derived by metabolism from glucose. RESULTS: No stimulation of chondroitin (35)S-sulfate synthesis was found at concentrations of glucosamine up to 1 mM, a concentration of (3)H-glucosamine well above the concentrations that could be presented to cartilage after ingestion of advertised amounts of glucosamine. Furthermore, there was significant dilution of exogenous glucosamine by endogenous glucosamine provided by metabolism from glucose. CONCLUSION: The results indicate that exogenous glucosamine does not stimulate chondroitin sulfate synthesis by human chondrocytes. Furthermore, the cells have the capacity to form amounts of glucosamine from glucose far in excess of that provided from exogenous sources, except at concentrations greater than could possibly be achieved with oral administration of glucosamine.

Cell Line, Transformed↗
PubMed1986

Thrombin inhibitory activity of heparin cofactor II depends on the molecular weight and sulfate amount of dextran sulfate.

The effect of molecular weight and sulfate amount of sulfated polysaccharide on the thrombin inhibitory activity of heparin cofactor II was investigated by using various dextran sulfate fractions with different molecular weight and sulfur content. The activity of dextran sulfate fractions of each size increased as the sulfur content was increased from 9 to 18%, and the activity decreased in molecules below 10 kDa. The maximum second order rate constant of heparin cofactor II-thrombin reaction in the presence of the fractions of over-10 kDa and 18% sulfur was 2.7 X 10(8) M-1 min-1 that was almost same as in the presence of heparin or dermatan sulfate. On the other hand, dextran sulfate accelerated antithrombin III-thrombin reaction only about 40-fold less than heparin. These results indicate that a large molecular size and significant amount of sulfate groups are only essential in the acceleration of the thrombin inhibitory activity of heparin cofactor II, whereas a specific sequence of heparin is required to that of antithrombin III.

Animals↗
PubMed1998

Glycosaminoglycan sulfation in human osteoarthritis. Disease-related alterations at the non-reducing termini of chondroitin and dermatan sulfate.

Chondroitin lyase products of aggrecan and small proteoglycans from normal and osteoarthritic cartilages were analyzed for chain internal Deltadisaccharides and terminal mono- or disaccharides. Chondroitin and dermatan sulfate chains from arthritic cartilages were of essentially normal size and internal sulfation but had significantly altered sulfation of the terminal residues. Whereas in normal cartilage, approximately 60% of terminal GalNAc4S was 4, 6-disulfated, it was reduced to approximately 30% in osteoarthritic cartilage. This is most likely due to a lower terminal GalNAc4, 6S-disulfotransferase activity and reveals that metabolic changes in osteoarthritis can affect this distinct sulfation step during chondroitin and dermatan sulfate synthesis. GlcAbeta1,3GalNAc6S-, the mimotope for antibody 3B3(-), was present on approximately 8 and approximately 10% of chains from normal and osteoarthritic cartilages, respectively. 3B3(-) assayed by immunodot blot was within the normal range for most osteoarthritic samples, with only 5 of 24 displaying elevated reactivity. This resulted not from a higher content of mimotope, but possibly from other structural changes in the proteoglycan that increase mimotope reactivity. In summary, chemical determination of sulfation isomers at the non-reducing termini of chondroitin and dermatan sulfate provides a reliable assay for monitoring proteoglycan metabolism not only during normal growth of cartilage but also during remodeling of cartilage in osteoarthritis.

Adult↗
PubMed1976

Dermatan sulfate-chondroitin sulfate copolymers from ambilical cord. Isolation and characterization.

Dermatan sulfate-chondroitin sulfate copolymers have been isolated from human umbilical cord as a major galactosaminoglycan component of this tissue. The galactosaminoglycan fraction was obtained from this tissue by papain [EC 3.4.22.2] digestion followed by precipitation with cetylpyridinium chloride in a yield of 700 mg per 100 g of dry tissue. Ethanol fractionation resolved 4-5 subfractions differing in relative content of L-iduronic acid and D-glucuronic acid. No galactosaminoglycan containing either solely L-iduronic acid or D-glucuronic acid was obtained. The copolymeric structure of the material in each subfraction was demonstrated by analysis of oligosaccharide fragments obtained by chondroitinase-AC [EC 4.2.2.5] digestion. All the polymers contained repeating disaccharide units, D-glucuronosyl-N-acetylgalactosamine, D-glucuronosyl-N-acetylgalactosamine 4-sulfate, D-glucuronosyl-N-acetyl-galactosamine 6-sulfate, and L-iduronosyl-N-acetylgalactosamine 4-sulfate, of which D-glucuronosyl-N-acetylgalactosamine 6-sulfate and L-iduronosyl-N-acetylgalactosamine 4-sulfate were predominant. Both iduronic acid- and glucuronic acid-containing units were arranged in clusters. The presence of a considerable amount of nonsulfated disaccharide units was noted. The copolymers show extensive polydispersity in electrophoresis on cellulose acetate and gel chromatography on Sephadex G-200.

Amino Acids↗
PubMed1979

The effect of aging on the synthesis of hexosamine-containing substances from rat costal cartilage. A decrease in sulfation of chondroitin sulfate with aging.

The amount of glycosaminoglycan (GAG) in dry costal cartilage tissue of rats decreased with aging, while the GAG content in mg DNA (unit cartilage cell) remained the same with aging. These results can be explained by the finding that the total number of cartilage cells decreased with aging. Electrophoretic analysis showed that chondroitin 4-sulfate was the major GAG in rat costal cartilage of various ages. Rat costal cartilage of different ages was incubated with radioactive precursors, and newly synthesized GAG was prepared and the radioactivity analyzed to determine the biosynthetic activity. As to changes in the radioactivity uptake with aging per mg dry cartilage tissue, aging influenced [35S]sulfate incorporation into GAG more significantly than [3H]glucosamine incorporation into GAG. There was a significant decrease in the specific radioactivity of [35S]sulfate per mg DNA (unit cartilage cell), whereas the specific radioactivity of [3H]glucosamine per mg DNA did not change significantly with aging. Both the total sulfotransferase activity and the specific activity per mg DNA decreased significantly with aging. Analysis of disaccharide units formed after chondroitinase ABC digestion of labeled GAG isolated from young and old cartilage showed that the percentage of incorporation of [3H]glucosamine into deltaDi-OS increased significantly with aging. These results suggested that the appearance of nonsulfated positions in the structure of the chondroitin sulfate chain increased with aging. On the basis of gel chromatography on Bio-Gel A-1.5 m no significant difference in the approximate molecular size of chondroitin sulfate was observed between the young and old GAG samples. The present study indicated that the sulfation of chondroitin sulfate chains from rat costal cartilage decreased with the process of aging.

Aging↗
PubMed1989

Immunohistochemical localization of chondroitin sulfate and dermatan sulfate proteoglycan in human gingival connective tissue.

This study investigated the immunohistochemical localization of chondroitin sulfate (chondroitin, 4-sulfate and 6-sulfate) and dermatan sulfate proteoglycan (PG) in human gingival connective tissue, using monoclonal antibodies. Dermatan sulfate was found to be widespread in connective tissue, with an especially strong response shown in collagen fiber bundles under the epithelial basement membrane. Chondroitin 4-sulfate occurred widely in connective tissue but showed only a weak response. Chondroitin 6-sulfate was located in peripheral blood vessels. Chondroitin was not detected in gingival connective tissue.

Antibodies, Monoclonal↗
PubMed1994

Interaction of hepatocyte growth factor with heparan sulfate. Elucidation of the major heparan sulfate structural determinants.

We have demonstrated by affinity chromatography that hepatocyte growth factor (HGF) binds strongly to heparan sulfate (HS). This substantiates previous suggestions that cell-surface heparan sulfate proteoglycans constitute the so-called low affinity cellular binding sites for HGF. Using a recombinant human HGF affinity column, we have analyzed the effects of various specific chemical and enzymatic modifications/depolymerizations of HS on its affinity in order to elucidate the polysaccharide structural determinants. Interaction is shown to be only slightly affected by digestion with heparinase I or III or by replacement of N-sulfates with N-acetyl groups. This suggests a specific role for sulfated domains containing nonsulfated IdceA residues, with only a small contribution from N-sulfates and IdceA(2-OSO3) residues. In addition, disaccharide analyses of various HGF-binding oligosaccharides indicate that affinity is more closely associated with 6-O-sulfation of GlcNSO3 residues than with sulfation at any other position. Although interaction can be demonstrated with heparinase III-resistant oligosaccharides as small as hexasaccharides, the highest affinity was found with oligosaccharides containing a minimum of 10-12 monosaccharides. The structural specificity of the HGF-HS interaction is thus shown to be radically different from that previously described for the basic fibroblast growth factor-HS interaction.

Animals↗
PubMed1987

Modulation of heparan sulfate biosynthesis. Effects of 6-diazo-5-oxo-L-norleucine and low glutamine on the synthesis of heparan sulfate proteoglycan by human colon carcinoma cells.

We have investigated the biosynthesis of heparan sulfate proteoglycan in human colon carcinoma cells cultured in either the absence of L-glutamine or the presence of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue. Following a 24-h incubation with 100 micrograms/ml DON (0.58 mM) or without L-glutamine, the incorporation of [35S]sulfate was maximally inhibited to about 50%, whereas the incorporation of [3H]leucine or [3H]serine and their specific activity were not significantly affected. Several lines of evidence indicate that the inhibition of [35S]sulfate incorporation was mediated by a reduction in the intracellular pool of UDP-N-acetylhexosamine: the intracellular hexosamine levels were reduced by about 50%; taking into account the changes in specific activity, the incorporation of [3H]glucosamine was also significantly inhibited; and exogenous D-glucosamine (2.3 mM) was capable of substantially reversing the inhibitory effects of DON. This decrease in endogenous sugar supply resulted in the generation of an underglycosylated, lower buoyant density proteoglycan which contained significantly fewer heparan sulfate chains of otherwise normal size and sulfation and even fewer O-linked oligosaccharides. These biochemical changes were corroborated ultrastructurally by the appearance of smaller ruthenium red-stained proteoglycans on the surface of the cells. DON also caused a marked inhibition of cell proliferation and profound morphological changes, both of which were reversible upon culturing in DON-free medium. These results demonstrate that perturbations in glutamine metabolism have profound effects on the structure of heparan sulfate proteoglycan and on the phenotype of human colon carcinoma cells and indicate that DON treatment could be useful for studying post-translational modifications of proteoglycans in various cell systems.

Azo Compounds↗
PubMed2002

Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate.

Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed.

Bacterial Proteins↗
PubMed1985

Contraluminal sulfate transport in the proximal tubule of the rat kidney. II. Specificity: sulfate-ester, sulfonates and amino sulfonates.

In order to study the specificity for the contraluminal sulfate transport system the inhibitory potency of sulfate esters and sulfonate compounds on the 35SO4(2-) influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: 1. From 10 sulfate monoesters tested 9 inhibited contraluminal sulfate influx with an app. Ki between 0.6 and 6 mmol/l; the two sulfate diesters tested, however, did not. 2. Out of 8 aliphatic sulfonate compounds only three, having a NH- or OH-group in a suitable position, exerted a moderate inhibition (app. Ki ca. 2-6 mmol/l). 3. Amongst 14 benzene sulfonates tested only 2 compounds (5-nitrobenzene-sulfonate and 2-hydroxy-5-nitrobenzenesulfonate) inhibited with a Ki less than 5 mmol/l. 4. Out of 10 naphthalene sulfonates tested 8 inhibited with a Ki less than 5; the highest inhibition was seen with the NH-containing 8-anilinonaphthalene-1-sulfonate (ANS), but no inhibition with 2 compounds containing an amino group. 5. From the polycyclic sulfonates pyrene-3-sulfonate and anthracene-1-sulfonate inhibited with a Ki of approximately 2 mmol/l, while no inhibition was seen with anthracene-2-sulfonate. 6. Out of 4 amino-sulfonates tested benzene-1-amino-sulfonate and a similar benzyl-analog inhibited with a Ki of 1 mmol/l and smaller; cyclohexyl-1-amino-sulfonate (cyclamate), however, inhibited only slightly (app. Ki of 6 mmol/l). The data indicate that sulfate monoesters are well accepted by the contraluminal sulfate transport system. The affinity of sulfonate compounds to this system depends on neighbouring OH-groups --NH-groups, meta-positioned electronegative groups or a hydrophobic moiety in an appropriate position.

Animals↗
PubMed1990

Dermatan sulfate and de-sulfated heparin solubilized collagen-tailed acetylcholinesterase from the rat neuromuscular junction.

We are interested in the study of the interactions involved in the attachment of collagen-tailed acetylcholinesterase (AChE) to the synaptic basal lamina. The fact that AChE occupies less than 0.1% of the muscle basal lamina, suggests that there is a very high specificity in the interaction that defines its distribution. We have previously found that asymmetric AChE is bound to the neuromuscular junction via heparan sulfate proteoglycans. Sulfated glycosaminoglycans as heparan sulfate and heparin extracted the asymmetric AChE from the synaptic basal lamina. Here we show that dermatan sulfate as well as de-sulfated heparin, are also able to extract collagen-tailed AChE. Taking into account that the solubilization of the asymmetric AChE is concomitant with the liberation of a dermatan sulfate proteoglycan from the rat neuromuscular junction, the present results open the possibility that the collagen-tailed AChE is also anchored to dermatan sulfate proteoglycans at the synaptic basal lamina.

Acetylcholinesterase↗
PubMed1976

On the structure of heparitin sulfates. Analyses of the products formed from heparitin sulfates by two heparitinases and a heparinase from Flavobacterium heparinum.

The analyses of the products formed from heparitin sulfates by the action of two heparitinases and a heparinase from Flavorbacterium heparinum is reported. Heparitin sulfates A and B are degraded by heparitinase I yielding two disaccharides, one of them composed of N-acetylucosamine and an unsaturated uronic, joined by alpha(1 lead to 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 alpha(1 lead to 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 alpha-glycuronidase from Flavobacterium heparinum yielding the respective monosaccharides. Based on these and other analyses the possible structures of the heparitin sulfates are proposed.

Acetylglucosamine↗
PubMed1977

Release of O-sulfate groups under mild acid hydrolysis conditions used for estimation of N-sulfate content.

The treatment of chondroitin sulfate isolated from cultured B16 mouse melanoma cells with 0.04 M HCl at 100 degrees C for 90 min released up to 45% of O-sulfate residues as free inorganic sulfate. In addition to the release of inorganic sulfate, extensive degradation of this polysaccharide as well as of cartilage chondroitin sulfate, pig rib cartilage proteoglycan, heparin and hyaluronic acid was also evident under these conditions. The above hydrolysis conditions are used for characterizing 35S-labeled heparan sulfates synthesized by cultured cells and to calculate ratio of N- and O-sulfates in these molecules. Our results suggest that caution is necessary in interpreting the results of mild acid hydrolysis of glycosaminoglycans.

Animals↗