Deaminative Cleavage Research Articles

Structural differences between beef-lung heparan sulphates with specific self-associations

Three, specifically self-associating variants of heparan sulphate (HS2-A, HS3-A, and HS4-A) from beef lung were subjected to ( a) deaminative cleavage of bonds between 2-deoxy-2-sulphoaminoglucose and uronic acid and ( b) periodate oxidation of glucuronic acid residues in fully N-acetylated block-regions. In addition, the periodate-oxidised and alkali-cleaved chains were re-oxidised with periodate to identify the glucuronic acid residues in the N-sulphate-containing regions. The results showed that HS2-A was distinguished by much longer (GlcA-GlcNAc) n -segments than HS3-A and HS4-A. The latter two species were characterised by the structure of the variously N-acetyl- and N-sulphate-containing regions. In HS3-A, there was a significant contribution from segments composed of both N-acetylated and N-sulphated 2-amino-2-deoxyglucose residues. The N-sulphate-rich regions contained chiefly iduronic acid. In contrast, HS4-A had mixed or alternating arrangements of the two epimeric uronic acids in the N-sulphate-rich regions. These differences may be the basis for specific self-associations between heparan sulphate chains.

The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence. Chemical and 13C nuclear-magnetic-resonance studies.

The chemical composition and the 13C n.m.r. spectra of heparin oligosaccharides (essentially octasaccharides), having high affinity for antithrombin III and high anti-(Factor Xa) activity, prepared by three independent approaches (extraction, partial deaminative cleavage with HNO2 and partial depolymerization with bacterial heparinase), leading to different terminal residues, have been studied and compared with those of the corresponding inactive species. Combined wit chemical data, the spectra of the active oligosaccharides and of their fragmentation products afforded information on composition and sequence. The three types of active oligosaccharides were shown to have the common hexasaccharide core I-Aa-G-As*-Is-As, where I and alpha-L-idopyranosyl-uronic acid, Aa = 2-acetamido-2-deoxy-alpha-D-glucopyranose, G = beta-D-glucopyranosyl-uronic acid, Is = alpha-L-idopyranosyluronic acid 2-O-sulphate, As = 2-deoxy-2-sulphamino-alpha-D-glucopyranose 6-O-sulphate. The fourth residue (As*) is an unusually substituted amino sugar resistant to mild deamination. The 13C spectra of the active species are characterized by signals from the above atypical amino sugar, the most evident of which is at 57.7 p.p.m. These signals, compared with those of appropriate synthetic model compounds, are compatible with the recently proposed 3-O-sulphation of the residue As* [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555].

Cells selected for high tumorigenicity or transformed by simian virus 40 synthesize heparan sulfate with reduced degree of sulfation.

Cell lines, selected from two independent clones of an established mouse embryo cell line by their ability to grow as solid tumors in immunocompetent syngeneic hosts, were found to have the same alteration in anion exchange properties as was previously reported for simian virus 40 (SV40)-transformed subclones. One tumor cell line (219CT) and one SV40-transformed subclone (215CSC) were selected for further detailed comparison with their common parent clone (210C). Cellulose acetate electrophoresis at pH 1.0 showed that 215CSC heparan sulfate had a slight overall decrease in sulfation compared with heparan sulfate from 210C; however, no gross difference in sulfation could be detected between heparan sulfate from 219CT and 210C. Analysis of the products of deaminative cleavage of heparan sulfate by nitrous acid under conditions where cleavage occurs quantitatively at N-sulfated glucosamine residues showed that, although heparan sulfate from the three cell lines gave similar yields of O-sulfated disaccharides, both 215CSC and 219CT had only about half as many O-sulfate residues in higher molecular weight oligosaccharides compared to heparan sulfate from 210C. Enzymatic degradation of heparan sulfate with a mixture of enzymes from Flavobacterium heparinum showed that this common alteration in heparan sulfate from both 215CSC and 219CT resulted from a 30% decrease in glucosamine residues bearing 6-O-sulfate groups. As this decrease in 6-O-sulfate glucosamine residues occurs in regions of the chain containing relatively few sulfate groups, it is clear that certain sequences of charged groups present in heparan sulfate frm 210C will be found only rarely in heparan sulfate from 215CSC and 219CT. It is suggested that this will result in alterations of the interaction of heparan sulfate with other molecules in the microenvironment at the cell surface which may be important in the control of such phenomena as cell growth and adhesion.

Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin.

An octasaccharide with high affinity for antithrombin was isolated after partial deaminative cleavage of heparin with nitrous acid. After conversion of the 2,5-anhydro-D-mannose end group to anhydro[1-3H]mannitol, labeled pentasaccharide was released from the octasaccharide by periodate-alkali treatment. Incubation of the pentasaccharide with a recently discovered 3,O-sulfatase from human urine resulted in desulfation, suggesting the occurrence of a 3-sulfate group on the terminal glucosamine residue. The same glucosamine residue was recovered as a 2,5-anhydro[1-3H]mannitol derivative by a procedure involving deamination of the octasaccharide with nitrous acid, reduction of the products with sodium boro[3H]hydride, isolation of 3H-labeled tetrasaccharide by gel chromatography, and release of the labeled end-group by periodate-alkali treatment. Paper electrophoresis indicated disulfated anhydro[3H]mannitol, presumably sulfated at C3 and C6, as a major component, along with smaller amounts of monosulfated (presumably 3-sulfated) anhydro[3H]mannitol. Similar treatment of an analogous tetrasaccharide derived from heparin with low affinity for antithrombin failed to produce any disulfated anhydromannitol. These results suggest that 3-sulfated glucosamine is a unique component of high-affinity heparin, located at a specific position in the antithrombin-binding sequence of the molecule.

Interaction between heparan sulphate chains. II. Structural characterization of iduronate- and glucuronate-containing sequences in aggregating chains.

Abstract A heparan sulphate fraction (uronic acid composition: 20% sulphated iduronate, 15% iduronate and 65% glucuronate of total uronate) was separated into aggregating and non-aggregating chains by gel chromatography. 13 C-NMR analyses revealed that non-aggregating chains had a higher degree of sulphation than did aggregating chains. In aggregating chains, there was more N -acetyl-glucosamine than N -sulphamidoglucosamine; the extent of C-6 sulphation of the latter moiety was low and most of the iduronate residues were non-sulphated. In non-aggregating chains, the N -acetyl-to- N -sulphate ratio was approx. 2 : 1, the N -sulphated glucosamines were also largely C-6 sulphated and the sulphated iduronates were concentrated to these species. Both preparations were subjected to deaminative cleavage which produces fragments like uronate-( N -acetylglucosamine-uronate) n -anhydromannose. Tetrasaccharides ( n = 1) were further fractionated into non-, mono-, di- and trisulphated species by ion-exchange chromatography. The tetrasaccharides have the general carbohydrate structure uronate- N -acetylglucosamine-glucuronate-anhydromannose. Non-reducing terminal glucuronate was removed by β-glucuronidase. The results showed that saccharides containing glucuronate in both positions were more prevalent in the products of aggregating chains. The β-glucuronidase-resistant saccharides (carrying either sulphated or non-sulphated iduronate in non-reducing terminal position) were oxidised with periodate under conditions where non-sulphated residues are degraded, whereas sulphated residues are resistant. Mono-sulphated and di-sulphated tetrasaccharides from aggregating chains were extensively degraded indicating that iduronate- N -acetylglucosamine-glucuronate-anhydromannose was the major sequence. In saccharides from non-aggregating chains iduronate was frequently sulphated. The results of this and previous investigations (Fransson, L.-A., Nieduszynski, I.A. and Sheehan, J.K. (1980) Biochim. Biophys. Acta 630, 287–300) indicate that an alternating arrangement of iduronate and glucuronate in aggregating chains is present both in N -sulphated block regions and in regionsthat carry alternating N -acetyl- and N -sulphated glucosamine.

Structural studies on heparan sulphate from human lung fibroblasts. Characterization of oligosaccharides obtained by selective periodate oxidation of D-glucuronic acid residues followed by scission in alkali.

1. (3)H- and (35)S-labelled heparan sulphate was isolated from monolayers of human lung fibroblasts and subjected to degradations by (a) deaminative cleavage and (b) periodate oxidation/alkaline elimination. Fragments were resolved by gel- and ion-exchange-chromatography. 2. Deaminative cleavage of the radioactive glycan afforded mainly disaccharides with a low content of ester-sulphate and free sulphate, indicating that a large part (approx. 80%) of the repeating units consisted of uronosyl-glucosamine-N-sulphate. Blocks of non-sulphated [glucuronosyl-N-acetyl glucosamine] repeats (3-4 consecutive units) accounted for the remainder of the chains. 3. By selective oxidation of glucuronic acid residues associated with N-acetylglucosamine, followed by scission in alkali, the radioactive glycan was degraded into a series of fragments. The glucuronosyl-N-acetylglucosamine-containing block regions yielded a compound N-acetylglucosamine-R, where R is the remnant of an oxidized and degraded glucuronic acid. Periodate-insensitive uronic acid residues were recovered in saccharides of the general structure glucosamine-(uronic acid-glucosamine)(n)-R. 4. Further degradations of these saccharides via deaminative cleavage and re-oxidations with periodate revealed that iduronic acid may be located in sequences such as glucosamine-N-sulphate-->iduronic acid-->N-acetylglucosamine. Occasionally the iduronic acid was sulphated. Blocks of iduronic acid-containing repeats may contain up to five consecutive units. Alternating arrangements of iduronic acid- and glucuronic acid-containing repeats were also observed. 5. (3)H- and (35)S-labelled heparan sulphates from sequential extracts of fibroblasts (medium, EDTA, trypsin digest, dithiothreitol extract, cell-soluble and cell-insoluble material) afforded similar profiles after both periodate oxidation/alkaline elimination and deaminative cleavage.

Heparan sulfates from Swiss mouse 3T3 and SV3T3 cells: O-sulfate difference.

A difference in the extent of sulfation between the heparan sulfate isolated from Swiss 3T3 mouse cells and that from Swiss 3T3 cells transformed by the DNA virus SV40 has been reported previously. This variance is manifested by different chromatographic and electrophoretic properties. Heparan sulfates from the two cell types were treated with nitrous acid under conditions that gave selective deaminative cleavage of glucosaminyl residues with sulfated amino groups in order to define the nature of the difference in sulfation further. The O-sulfate containing fragments from the heparan sulfates were compared by gel filtration and ion-exchange chromatography. The results showed that the 3T3 heparan sulfate contains 8% more O-sulfate than does the SV3T3 heparan sulfate. Analysis of uronic acids revealed that both types of heparan sulfates contain 45% L-iduronic acid and 55% D-glucuronic acid. These and other observations indicate that the primary difference in sulfation between the 3T3 and SV3T3 heparan sulfates lies in the extent of O-sulfation.

Structural Studies on Heparan Sulphates

Three heparan sulphate fractions were subjected to degradation by periodate oxidation and alkaline elimination. The starting materials were one low-sulphated fraction rich in glucuronic acid and N-acetylglucosamine (I), one fraction (IV) that was medium-sulphated, contained similar proportions of N-acetyl and N-sulphate as well as of glucuronic and iduronic acid and one fraction (V) that was oversulphated, contained an excess of N-sulphate and was rich in iduronic acid. Selective periodate oxidation of glucuronic acid residues within N-acetylated regions followed by scission in alkali yielded three categories of fragments that were isolated by gel and ion-exchange chromatography. (a) N-Acetylglucosamine-R compounds where R is the remnant of an oxidised and degraded glucuronic acid residue. (b) Medium-sized oligosaccharides, of the general structure glucosamine-(glycuronic acid-glucosamine)n-R where n = 1--4, contained largely glucuronic acid associated with glucosamines that were N-sulphated, N-acetylated or unsubstituted. In saccharides where n = 4 indications of an alternating arrangement of N-sulphate and N-acetyl groups as well as of iduronic and glucuronic acid residues were obtained. The glucuronic acid residues of the oligosaccharide fragments were susceptible to reoxidation with periodate. The smaller saccharides were not depolymerised by HNO2 despite the presence of N-sulphate groups. (c) Longer oligosaccharide fragments (n greater than or equal to 5) that were highly sulphated and contained heparin-like repeating units, i.e. iduronic acid--glucosamine-N-sulphate with ester-sulphate on both sugars. These oligosaccharides were susceptible to deaminative cleavage.

Structure of the antithrombin-binding site in heparin.

Heparin preparations from pig intestinal mucosa and from bovine lung were separated by chromatography on antithrombin-Sepharose into a high-affinity fraction (with high anticoagulant activity) and a low-affinity fraction (with low anticoagulant). Antithrombin-binding heparin fragments (12-16 monosaccharide units) were prepared, either by digesting a high-affinity heparin-antithrombin complex with bacterial heparinase or by partial deaminative cleavage of the unfractionated polysaccharide with nitrous acid followed by affinity chromatography on immobilized antithrombin. Compositional analysis based on separation and identification of deamination products reduced with sodium boro[3H]hydride showed that nonsulfated L-iduronic acid occurred in larger amounts in high-affinity heparin than in low-affinity heparin; furthermore, this component was concentrated in the antithrombin-binding regions of the high-affinity heparin molecules, amounting to approximately one residue per binding site. It is suggested that nonsulfated L-iduronic acid is essential for the anticoagulant activity of heparin. The location of the non-sulfated uronic acid in the antithrombin-binding site was determined by periodate oxidation of antithrombin-binding fragments containing a terminal 2,5-anhydro-D-[1-3H]mannitol unit. Tentative structures for antithrombin-binding sequences in heparin are proposed, including some structural variants believed to be compatible with, but not required for, activity.

α- l-Iduronidase, β- d-glucuronidase, and 2-sulfo- l-iduronate 2-sulfutase: preparation and characterization of radioactive substrates from heparin

Radioactive disaccharide substrates for α- l-iduronidase, β- d-glucuronidase, and 2-sulfo- l-iduronate 2-sulfatase have been prepared from heparin by deaminative cleavage followed by reduction with NaBT 4. Six disaccharides were isolated from this reaction mixture and identified. Acid hydrolysis of the major disaccharide, O-(α- l-idopyranosyluronic acid 2-sulfate)-(1→4)-(2,5-anhydro- d-mannitol- 1-t 6-sulfate (IdAs-Ms), produced 48% of O-(α- l-idopyranosyluronic acid)-(1→4)-(2,5-anhydro- d-mannitol- 1-t 6-sulfate) (IdA-Ms) and 25% of O-(α- l-idopyranosyluronic acid)-(1→4)-2,5-anhydro- d-mannitol- 1-t. The most-sensitive substrate for determining a- l-iduronidase activity was IdA-Ms which, when incubated with leucocyte and skin-fibroblast homogenates prepared from patients having a deficiency of a- l-iduronidase (Mucopolysaccharidosis Type I; MPS-I), was hydrolysed to yield 2,5-anhydro- d-mannitol- 1-t 6-sulfate at a rate 50-times less than that found for normal control-preparations. Similarly, O-(β- d-glucopyranosyluronic acid)-(1→4)-(2,5-anhydro-D-mannitol- 1-t 6-sulfate) was degraded by whole-cell homogenates prepared from β- d-glucuronidase-deficient (Mucopolysaccharidosis, Type VII) fibroblasts, to yield 2,5-anhydro- d-mannitol- 1-t 6-sulfate at a rate 60-times less than that found for MPS-I and normal control-preparations. IdAs-Ms was degraded by 2-sulfo- l-iduronate 2-sulfatase at a rate more than 45-times greater than that found for O-( a- l-idopyranosyluronic acid 2-sulfate)-(1→4)-2,5-anhydro- d-mannitol- 1-t. C-6 Sulfation of the anhydro- d-mannitol- 1-t residue is an important structural determinant in the mechanism of action of both a- l-iduronidase and 2-sulfo- l-iduronate 2-sulfatase on disaccharide substrates.

Structure and metabolism of sulphated glycosaminoglycans in cultures of human fibroblasts. Structural characteristics of co-polymeric galactosaminoglycans in sequential extracts of fibroblasts during pulse-chase experiments

1. Human embryonic lung and skin fibroblasts were allowed to incorporate 32SO42- or 35SO42- and D-[1-3H]glucosamine. After removal of the medium the monolayer was subjected to sequential extractions by using EDTA, brief trypsin digestion, extraction with dithiothreitol ofllowed by freeze--thawing and extraction with trichloroacetic acid. The heparan sulphate and galactosaminoglycan contents of the various extracts were estimated after deaminative cleavage of the former component. Heparan sulphate was the major component of the trypsin digest, whereas galactosaminoglycans were the dominant component of other fractions. 2. Galactosaminoglycans of the various fractions were subjected to chemical (periodate oxidation/alkaline elimination) and enzymic (chondroitinase-AC and -ABC, as well as testicular hyaluronidase) degradations. Galactosaminoglycans from the insoluble cell fraction and the dithiothreitol extract contained larger amounts of L-iduronic acid than did those of other fractions. 3. Pulse-chase experiments were performed with and without replating of the cells at the start of the chase period. Radioactive glycans were isolated from the various extracts during the chase period. The half-lives of glycans of the insoluble cell fraction and the dithioreitol extract were shorter (5--8h) than were those of the trypsin digest and the EDTA extract (22h and 11h respectively). After replating of the cells in chase medium, radioactive cell-associated glycans were secreted from the cells and could be recovered in the trypsin digest, the EDTA extract and the medium. Furthermore, 35S/3H ratios of glycans from all these fractions decreased during the chase period. The following conclusions were reached. The insoluble cell fraction contains the synthesis pool and some structural material, whereas the soluble cell fraction is the storage and degradation pool. The dithiothreitol extract appears to contain the immediate precursors of secreted material. The trypsin-released glycans comprise structural components as well as material destined for pinocytosis or secretion into the medium. The EDTA extract is considered to consist of glycans en route to the medium. 4. The two presumptive precursor pools were preferentially depleted of L-iduronic acid-rich galactosaminoglycans during the chase. Glycans recovered from the trypsin digest, the EDTA extract and the medium during the chase contained larger amounts of periodate-resistant uronic acid residues (D-glucuronic acid and/or L-iduronic acid O-sulphate) than did their precursors. It is proposed that polymer-level modifications of secreted glycans are partly responsible for the results.

Synthesis and subcellular distribution of heparan sulfate in the rat exocrine pancreas

Tissue fragments and isolated acinar cells from adult rat pancreas rapidly incorporated inorganic 35SO 4 2− into macromolecules. Secretion of labeled macromolecules could be stimulated by the secretagogue carbamylcholine chloride. The principal sulfated product synthesized by isolated acinar cells was purified by diethylaminoethyl cellulose chromatography and was identified as heparan sulfate by digestion with purified heparitinase and by deaminative cleavage with nitrous acid. The results of subcellular fractionation of pancreas tissue labeled in vitro with 35SO 4 2− showed that sulfate-containing macromolecules were present both in soluble fractions and as tightly bound membrane components. Zymogen granules contained 14% of the total radioactivity incorporated; zymogen granule membranes, 3%; the soluble zymogen granule contents, 7%; microsomes, 30%; mitochondria, 14%; and the postmicrosomal supernate, 25%. Purified 35SO 4 2−-labeled zymogen granule membranes had the highest specific radioactivity of any fraction examined. Degradative analysis with nitrous acid indicated that the major sulfated product present in all of the subcellular fractions was heparan sulfate.

Galactosaminogalactan from cell walls of Aspergillus niger

A new heteropolysaccharide has been isolated by alkaline extraction of hyphal walls of Aspergillus niger NRRL 326 grown in surface culture. Its composition by weight, as determined by paper and gas chromatography and colorimetric analyses, is 70% galactose, 20% galactosamine, 6% glucose, and 1% acetyl. Two independent experiments have been used to ascertain copolymer structure: permeation chromatography in 6 M guanidinium hydrochloride, with controlled-pore glass columns of two fractionation ranges, and nitrous acid deaminative cleavage of galactosaminogalactan followed by reduction of fragments with [3H]borohydride and gel filtration chromatography. One of the tritiated fragments is tentatively identified as the disaccharide derivative galactopyranosyl 2,5-anhydrotalitol, on the basis of chromatographic properties and by kinetics of its acid hydrolysis. Smith degradation, methylation, deamination, and optical rotation studies indicate that the galactosaminogalactan consists of a linear array of hexopyranosyl units joined almost exclusively by alpha-(1 leads to 4) linkages. Hexosaminyl moieties are distributed randomly along the chains, which have an average degree of polymerization of about 100. The possible significance of this macromolecule in hyphal structure is considered.

Cleavage of macromolecular heparin by an enzyme from mouse mastocytoma.

Heparinase was isolated from a transplantable mouse mastocytoma, by salt extraction of a particulate fraction sedimenting at 20,000 times g, followed by precipitation from saturated ammonium sulfate. By use of gel chromatography through Sepharose 4B, the enzyme was shown to degrade macromolecular. 35S-labeled, mastocytomal heparin (K-av about 0.25) to products similar in size to commercial heparin (K-av about 0.85), apparently by nonrandom cleavage of a limited number of glycosidic linkages per molecule. Prolonged incubation times (up to 5 days, with repeated addition of enzyme) did not result in further degradation of the product. No significant depolymerizing activity was observed with any other glycosaminoglycan tested, including chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparan sulfate, and commercial heparin. The pH optimum for degradation of macromolecular heparin was around pH 5. The nature of the linkage cleaved by the heparinase was investigated by reduction of unlabeled polysaccharide degradation products with sodium [3H]borohydride. The degraded chains (but not the macromolecular substrate) incorporated significant amounts of tritium. An essentially monodisperse fraction of the labeled, degraded heparin was subjected to meniscus depletion sedimentation equilibrium ultracentrifugation, indicating a molecular weight of 14,500. By relating the molecular weight to the specific activity of the preparation, the amount of reducible groups was calculated to be approximately one per molecule. The 3H-labeled heparin was degraded to monosaccharides by a combination of acid hydrolysis and cleavage due to deamination with nitrous acid. Analysis of the degradation products, by paper electrophoresis and paper chromatography, showed a major radioactive component which behaved like L-gulonic acid. Since [3H]gulonic acid would be the expected reduction product of a polysaccharide molecule, containing a glucuronic acid residue in terminal position, these results tentatively suggest that the heparinase is an endoglucuronidase. By direct deaminative cleavage (no hydrolysis) of the 3H-labeled heparin, the glucosamine unit in penultimate position (i.e. adjacent to the [3H]gulonic acid residue) was shown to be 52% N-sulfated and 48% N-acetylated. As only 14% of the glucosamine was N-acetylated in the macromolecular heparin substrate, it is suggested that cleavage of this polysaccharide, by the heparinase, occurs in regions more abundant in N-acetylated glucosamine residues than other portions of the molecule. The possibility that formation and degradation of macromolecular heparin occurs also in mammalian species other than rodents in discussed.

Macromolecular properties and end-group analysis of heparin isolated from bovine liver capsule.

Glycosaminoglycans were extracted from bovine liver capsule with 4 M-guanidinium chloride, resulting in solubilization of approx. 90% of the total uronic acid-containing polysaccharide of the tissue. The extracted polysaccharide was purified and fractionated by anion-exchange chromatography on DEAE-cellulose, density-gradient ultracentrifugation in CsCl and finally gel chromatography on Sepharose 4B. By using these procedures, the two major polysaccharide components, dermatan sulphate and heparin, which constituted 55 and 30% respectively of the total glycosaminoglycan content of the tissue, were separated from each other. Analysis of the macromolecular properties of the two polysaccharides showed that heparin existed exclusively as single polysaccharide chains, whereas dermatan sulphate occurred largely as a proteoglycan (protein content, 74% dry wt.). The purified heparin preparation was subjected to sedimentation-equilibrium ultracentrifugation, indicating a molecular weight of 8800. Analysis for neutral sugars (by g.l.c.) showed 0.1 residue of xylose and 0.2 residue of galactose/polysaccharide chain; serine amounted to 0.3 residue/polysaccharide chain. Reduction of the heparin with NaB3H4 resulted in incorporation of 3H, approximately corresponding to one reducible group/polysaccharide chain. The 3H-labelled sugar residue was liberated by a combination of acid hydrolysis and deaminative cleavage of the polysaccharide with HNO2; it was subsequently identified as an aldonic acid by paper electrophoresis. Most of the heparin chains thus contained a uronic acid residue in reducing position. It is suggested that heparin isolated from bovine liver capsule is a degradation product released from larger molecules by an endo-glycuronidase.

An assay for iduronate sulfatase (hunter corrective factor)

A substrate for α- l-idopyranosyluronic acid 2-sulfate 2-sulfohydrolase (iduronate sulfatase), the enzyme deficient in the Hunter syndrome, was prepared by deaminative cleavage of heparin and subsequent reduction of the disulfated disaccharide fragment with sodium borotritide, to give O-(α- l-idopyranosyluronic acid 2-sulfate)-(1→4)-2,5-anhydro- d-[ 3H-1]mannitol 6-sulfate. Incubation of this compound with enzyme gave a monosulfated radioactive product, which can be separated from the substrate by paper chromatography or electrophoresis. The reaction follows Michaelis-Menten kinetics, with a K M of 3m m. The phosphate ion is a potent inhibitor.

Biosynthesis of Heparin: III. FORMATION OF IDURONIC ACID RESIDUES

Abstract Incubation of a microsomal fraction from mouse mastocytoma with UDP-[14C]glucuronic acid and unlabeled UDP-N-acetylglucosamine resulted in the formation of a nonsulfated polysaccharide containing [14C]glucuronic acid as the only labeled component present in significant amounts. Similar incubations, carried out in the presence of 3'-phosphoadenylylsulfate yielded a sulfated polymer, with [14C]iduronic acid comprising one-third of the total labeled uronic acid. Products obtained by sulfation of preformed, 14C-labeled glycosaminoglycan showed the same proportion of [14C]iduronic acid. Since the preformed polysaccharide contained radioactive glucuronic acid only, it is concluded that the [14C]iduronic acid residues of the sulfated product had been formed by C-5 epimerization of labeled glucuronic acid units, on the polymer level. Oligosaccharides, isolated after deaminative cleavage of 14C-labeled microsomal polysaccharide, were characterized with regard to the contents and location of iduronic acid and sulfate residues. The results showed that iduronic acid units and ester sulfate groups were accumulated within the same sections of the polysaccharide chain, and suggest therefore that the epimerization of glucuronic acid residues is somehow linked to the sulfation of hydroxyl groups.

Stoichiometry of the nitrous acid deaminative cleavage of model amino sugar glycosides and glycosaminoglycuronans.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTStoichiometry of the nitrous acid deaminative cleavage of model amino sugar glycosides and glycosaminoglycuronansJohn Ernest Shively and Harry E. ConradCite this: Biochemistry 1970, 9, 1, 33–43Publication Date (Print):January 6, 1970Publication History Published online1 May 2002Published inissue 6 January 1970https://doi.org/10.1021/bi00803a005RIGHTS & PERMISSIONSArticle Views300Altmetric-Citations98LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Epimerization of D-glucuronic acid to L-iduronic acid by deamination of heparins

Porcine α-heparin, whale ω-heparin and 4-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-D-glucuronic acid were deaminated with nitrous acid. Uronic acid in the deamination products was identified as L-iduronic acid by paper chromatography, gas-liquid chromatography, infrared spectrophotometry and carbazole-to-orcinol ratio of the color reaction. The present data indicate that the deaminative cleavage of the glycosidic linkage between C 4 of D-glucuronic acid and C 1 of D-glucosamine results in the concomitant epimerization of D-glucuronic acid to L-iduronic acid.

DEAMINATION OF THE PARTIALLY N-DEACETYLATED ACID MUCOPOLYSACCHARIDES.

1. The partially N-deacetylated preparations of sodium chondroitin sulfate A and of sodium hyaluronate were deaminated with nitrous acid. 2. The deamination products of the N-deacetylated hexosamines in these partially N-deacetylated mucopolysaccharides were shown to be hexoses and 2, 5-anhydrohexoses. The amounts of hexoses found in the non-dialyzable matters of the deamination products were more than twice as much as those of 2, 5-anhydrohexoses. 3. The experimental results indicate that the C3-substituted hexosaminidic linkages are, more or less, resistant against the deaminative cleavage to form 2, 5-anhydrohexoses with nitrous acid.